Immunomodulators

ABSTRACT

The present disclosure provides compounds which are immunomodulators and thus are useful for the amelioration of various diseases, including cancer and infectious diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 16/711,105, filed Dec. 11, 2019, which is a continuation of U.S.application Ser. No. 15/822,744, filed Nov. 27, 2017, issued as U.S.Pat. No. 10,633,419, which is a continuation of U.S. application Ser.No. 14/938,327 filed Nov. 11, 2015, issued as U.S. Pat. No. 9,856,292,which claims priority to U.S. Provisional Patent Application No.62/204,689, filed Aug. 13, 2015, now expired, U.S. Provisional PatentApplication No. 62/111,388, filed Feb. 3, 2015, now expired, and U.S.Provisional Patent Application No. 62/079,944, filed Nov. 14, 2014, nowexpired. The contents of all above-named applications are incorporatedherein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name 3338_1530006_SEQ_1; Size: 5853 bytes; and Date ofCreation: Apr. 11, 2022) filed with the application is incorporatedherein by reference in its entirety.

The present disclosure provides novel macrocyclic peptides which inhibitthe PD-1/PD-L1 and CD80/PD-L1 protein/protein interaction, and are thususeful for the amelioration of various diseases, including cancer andinfectious diseases.

The protein Programmed Death 1 (PD-1) is an inhibitory member of theCD28 family of receptors, that also includes CD28, CTLA-4, ICOS andBTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells(Agata et al., supra; Okazaki et al., Curr. Opin. Immunol., 14:779-782(2002); Bennett et al., J. Immunol., 170:711-718 (2003)).

The PD-1 protein is a 55 kDa type I transmembrane protein that is partof the Ig gene superfamily (Agata et al., Int. Immunol., 8:765-772(1996)). PD-1 contains a membrane proximal immunoreceptor tyrosineinhibitory motif (ITIM) and a membrane distal tyrosine-based switchmotif (ITSM) (Thomas, M. L., J. Exp. Med., 181:1953-1956 (1995); Vivier,E. et al., Immunol. Today, 18:286-291 (1997)). Although structurallysimilar to CTLA-4, PD-1 lacks the MYPPY motif that is critical for CD80CD86 (B7-2) binding. Two ligands for PD-1 have been identified, PD-L1(B7-H1) and PD-L2 (b7-DC). The activation of T cells expressing PD-1 hasbeen shown to be downregulated upon interaction with cells expressingPD-L1 or PD-L2 (Freeman et al., J. Exp. Med., 192:1027-1034 (2000);Latchman et al., Nat. Immunol., 2:261-268 (2001); Carter et al., Eur. JImmunol., 32:634-643 (2002)). Both PD-L1 and PD-L2 are B7 protein familymembers that bind to PD-1, but do not bind to other CD28 family members.The PD-L1 ligand is abundant in a variety of human cancers (Dong et al.,Nat. Med., 8:787-789 (2002)). The interaction between PD-1 and PD-L1results in a decrease in tumor infiltrating lymphocytes, a decrease inT-cell receptor mediated proliferation, and immune evasion by thecancerous cells (Dong et al., J. Mol. Med., 81:281-287 (2003); Blank etal., Cancer Immunol. Immunother., 54:307-314 (2005); Konishi et al.,Clin. Cancer Res., 10:5094-5100 (2004)). Immune suppression can bereversed by inhibiting the local interaction of PD-1 with PD-L1, and theeffect is additive when the interaction of PD-1 with PD-L2 is blocked aswell (Iwai et al., Proc. Natl. Acad. Sci. USA, 99:12293-12297 (2002);Brown et al., J. Immunol., 170:1257-1266 (2003)).

PD-L1 has also been shown to interact with CD80 (Butte M J et al,Immunity; 27:111-122 (2007)). The interaction PD-L1/CD80 on expressingimmune cells has been shown to be an inhibitory one. Blockade of thisinteraction has been shown to abrogate this inhibitory interaction(Paterson A M, et al., J Immunol., 187:1097-1105 (2011); Yang J, et al.J Immunol. August 1; 187(3):1113-9 (2011)).

When PD-1 expressing T cells contact cells expressing its ligands,functional activities in response to antigenic stimuli, includingproliferation, cytokine secretion, and cytotoxicity, are reduced.PD-1/PD-L1 or PD-L2 interactions down regulate immune responses duringresolution of an infection or tumor, or during the development of selftolerance (Keir, M. E. et al., Annu. Rev. Immunol., 26: Epub (2008)).Chronic antigen stimulation, such as that which occurs during tumordisease or chronic infections, results in T cells that express elevatedlevels of PD-1 and are dysfunctional with respect to activity towardsthe chronic antigen (reviewed in Kim et al., Curr. Opin. Imm. (2010)).This is termed “T cell exhaustion”. B cells also display PD-1/PD-ligandsuppression and “exhaustion”.

Blockade of PD-1/PD-L1 ligation using antibodies to PD-L1 has been shownto restore and augment T cell activation in many systems. Patients withadvanced cancer benefit from therapy with a monoclonal antibody to PD-L1(Brahmer et al., New Engl. J. Med. (2012)). Preclinical animal models oftumors and chronic infections have shown that blockade of the PD-1/PD-L1pathway by monoclonal antibodies can enhance the immune response andresult in tumor rejection or control of infection. Antitumorimmunotherapy via PD-1/PD-L1 blockade may augment therapeutic immuneresponse to a number of histologically distinct tumors (Dong, H. et al.,“B7-H1 pathway and its role in the evasion of tumor immunity”, J. Mol.Med., 81(5):281-287 (2003); Dong, H. et al., “Tumor-associated B7-H1promotes T-cell apoptosis: a potential mechanism of immune evasion”,Nat. Med., 8(8):793-800 (2002)).

Interference with the PD-1/PD-L1 interaction causes enhanced T cellactivity in systems with chronic infection. Blockade of PD-L1 causedimproved viral clearance and restored immunity in mice with chromoiclymphocytic chorio meningitis virus infection (Barber, D. L. et al.,“Restoring function in exhausted CD8 T cells during chronic viralinfection”, Nature, 439(7077):682-687 (2006)). Humanized mice infectedwith HIV-1 show enhanced protection against viremia and viral depletionof CD4+ T cells (Palmer et al., J. Immunol. (2013)). Blockade ofPD-1/PD-L1 through monoclonal antibodies to PD-L1 can restore in vitroantigen-specific functionality to T cells from HIV patients (Day, Nature(2006); Petrovas, J. Exp. Med. (2006); Trautman, Nature Med. (2006);D'Souza, J. Immunol. (2007); Zhang, Blood (2007); Kaufmann, Nature Imm.(2007); Kasu, J. Immunol. (2010); Porichis, Blood (2011)), HCV patients(Golden-Mason, J. Virol. (2007); Jeung, J. Leuk. Biol. (2007); Urbani,J. Hepatol. (2008); Nakamoto, PLoS Path. (2009); Nakamoto,Gastroenterology (2008)) and HBV patients (Boni, J. Virol. (2007);Fisicaro, Gastro. (2010); Fisicaro et al., Gastroenterology (2012); Boniet al., Gastro. (2012); Penna et al., J. Hep. (2012); Raziorrough,Hepatology (2009); Liang, World J. Gastro. (2010); Zhang, Gastro.(2008)).

Blockade of the PD-L1/CD80 interaction has also been shown to stimulateimmunity (Yang J., et al., J Immunol. August 1; 187(3):1113-9 (2011)).Immune stimulation resulting from blockade of the PD-L1/CD80 interactionhas been shown to be enhanced through combination with blockade offurther PD-1/PD-L1 or PD-1/PD-L2 interactions.

Alterations in immune cell phenotypes are hypothesized to be animportant factor in septic shock (Hotchkiss, et al., Nat Rev Immunol(2013)). These include increased levels of PD-1 and PD-L1 (Guignant, etal, Crit. Care (2011)), Cells from septic shock patients with increasedlevels of PD-1 and PD-L1 exhibit an increased level of T cell apoptosis.Antibodies directed to PD-L1, can reduce the level of Immune cellapoptosis (Zhang et al, Crit. Care (2011)). Furthermore, mice lackingPD-1 expression are more resistant to septic shock symptoms thanwildtype mice. Yang J., et al. J Immunol. August 1; 187(3):1113-9(2011)). Studies have revealed that blockade of the interactions ofPD-L1 using antibodies can suppress inappropriate immune responses andameliorate disease signs.

In addition to enhancing immunologic responses to chronic antigens,blockade of the PD-1/PD-L1 pathway has also been shown to enhanceresponses to vaccination, including therapeutic vaccination in thecontext of chronic infection (Ha, S. J. et al., “Enhancing therapeuticvaccination by blocking PD-1-mediated inhibitory signals during chronicinfection”, J Exp. Med., 205(3):543-555 (2008); Finnefrock, A. C. etal., “PD-1 blockade in rhesus macaques: impact on chronic infection andprophylactic vaccination”, J. Immunol., 182(2):980-987 (2009); Song,M.-Y. et al., “Enhancement of vaccine-induced primary and memoryCD8+t-cell responses by soluble PD-1”, J. Immunother., 34(3):297-306(2011)).

The molecules described herein demonstrate the ability to block theinteraction of PD-L1 with PD-1, in both biochemical and cell-basedexperimental systems. These results are consistent with a potential fortherapeutic administration to enhance immunity in cancer or chronicinfection, including therapeutic vaccine.

The macrocyclic peptides described herein are capable of inhibiting theinteraction of PD-L1 with PD-1 and with CD80. These compounds havedemonstrated highly efficacious binding to PD-L1, blockade of theinteraction of PD-L1 with either PD-1 or CD80, and are capable ofpromoting enhanced T cell functional activity, thus making themcandidates for parenteral, oral, pulmonary, nasal, buccal and sustainedrelease formulations.

In its first embodiment the present disclosure provides a compound offormula (I)

or a pharmaceutically acceptable salt thereof, wherein:

A is selected from

wherein:

denotes the point of attachment to the carbonyl group and

denotes the point of attachment to the nitrogen atom;

n is 0 or 1;

m is 1 or 2;

m′ is 0 or 1;

w is 0, 1, or 2;

R^(x) is selected from hydrogen, amino, hydroxy, and methyl;

R¹⁴ and R¹⁵ are independently selected from hydrogen and methyl;

R^(16a) is selected from hydrogen and C₁-C₆ alkyl;

R¹⁶ is selected from

—(C(R^(17a))₂)₂—X—R³⁰,

—C(R^(17a))₂C(O)N(R^(16a))C(R^(17a))₂—X′—R³¹,

—C(R^(17a))₂[C(O)N(R^(16a))C(R^(17a))₂]_(w′)—X—R³¹,

—(C(R^(17a))(R¹⁷)C(O)NR^(16a))_(n′)—H; and

—(C(R^(17a))(R¹⁷)C(O)NR^(16a))_(m′—C(R) ^(17a))(R¹⁷)—CO₂H;

wherein:

w′ is 2 or 3;

n′ is 1-6;

m′ is 0-5;

X is a chain of between 1 and 172 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two,three, or four groups selected from —NHC(O)NH—, and —C(O)NH— embeddedtherein; and wherein the chain is optionally substituted with one to sixgroups independently selected from —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and—(CH₂)CO₂H;

X′ is a chain of between 1 and 172 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two,three, or four groups selected from —NHC(O)NH—, and —C(O)NH— embeddedtherein; and wherein the chain is optionally substituted with one to sixgroups independently selected from —CO₂H, —C(O)NH₂, and —CH₂CO₂H,provided that X′ is other than unsubstituted PEG;

R³⁰ is selected from —CO₂H, —C(O)NR^(w)R^(x), and —CH₃ herein R^(w) andR^(x) are independently selected from hydrogen and C₁-C₆alkyl, providedthat when X is all carbon, R³⁰ is other than —CH₃;

R³¹ is —CO₂H, —C(O)NR^(w)R^(x), —CH₃, alexa-5-SDP, and biotin;

each R^(17a) is independently selected from hydrogen, C₁-C₆alkyl,—CH₂OH, —CH₂CO₂H, —(CH₂)₂CO₂H,

each R¹⁷ is independently selected from hydrogen, —CH₃, (CH₂)_(z)N₃,—(CH₂)_(z)NH₂, —X—R³¹, —(CH₂)_(z)CO₂H, —CH₂OH, CH₂C═CH, and—(CH₂)_(z)-triazolyl-X—R³⁵, wherein z is 1-6 and R³⁵ is selected from—CO₂H, —C(O)NR^(w)R^(x), CH₃, biotin, -2-fluoropyridine,—C(O)—(CH₂)₂—C(O)O-vitamin E, —C(O)O-vitamin E; and

provided at least one R¹⁷ is other than hydrogen, —CH₃, or —CH₂OH;

R^(c), R^(f), R^(h), R^(i), R^(m), and R^(n) are hydrogen;

R^(a), R^(e), R^(j), and R^(k), are each independently selected fromhydrogen and methyl;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³ areindependently selected from a natural amino acid side chain and anunnatural amino acid side chain or form a ring with the correspondingvicinal R group as described below;

R^(e) and R^(k) can each form a ring with the corresponding vicinal Rgroup and the atoms to which they are attached selected from azetidine,pyrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole;wherein each ring is optionally substituted with one to four groupsindependently selected from amino, cyano, methyl, halo, and hydroxy;

R^(b) is methyl or, R^(b) and R², together with the atoms to which theyare attached, form a ring selected from azetidine, pyrolidine,morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein eachring is optionally substituted with one to four groups independentlyselected from amino, cyano, methyl, halo, and hydroxy;

R^(d) is hydrogen or methyl, or, R^(d) and R⁴, together with the atomsto which they are attached, can form a ring selected from azetidine,pyrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole;wherein each ring is optionally substituted with one to four groupsindependently selected from amino, cyano, methyl, halo, hydroxy, andphenyl;

R^(g) is hydrogen or methyl or R^(g) and R⁷, together with the atoms towhich they are attached, can form a ring selected from azetidine,pyrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole;wherein each ring is optionally substituted with one to four groupsindependently selected from amino, benzyl optionally substituted with ahalo group, benzyloxy, cyano, cyclohexyl, methyl, halo, hydroxy,isoquinolinyloxy optionally substituted with a methoxy group,quinolinyloxy optionally substituted with a halo group, and tetrazolyl;and wherein the pyrrolidine and the piperidine ring are optionally fusedto a cyclohexyl, phenyl, or indole group; and

R¹ is methyl or, R¹ and R¹², together with the atoms to which they areattached, form a ring selected from azetidine and pyrolidine, whereineach ring is optionally substituted with one to four independentlyselected from amino, cyano, methyl, halo, and hydroxy.

In a first aspect of the first embodiment the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein A is

In a second aspect of the first embodiment:

m and w are 1; and

R¹⁴, R¹⁵, and R^(16a) are each hydrogen.

In a third aspect of the first embodiment:

R¹⁶ is —(C(R^(17a))₂)₂—X—R³⁰.

In a fourth aspect of the first embodiment:

each R^(17a) is hydrogen;

X is a chain of between 8 and 46 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two, orthree C(O)NH groups embedded therein; and wherein the chain isoptionally substituted with one or two groups independently selectedfrom —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and —CH₂CO₂H; and

R³⁰ is selected from —CH₃, —CO₂H, and —C(O)NH₂; provided that when X isall carbon, R³⁰ is other than —CH₃.

In a fifth aspect of the first embodiment the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein:

A is

m and w are 1;

R¹⁴, R¹⁵, and R^(16a) are each hydrogen; and

R¹⁶ is —C(R^(17a))₂C(O)N(R^(16a))C(R^(17a))₂—X′—R³¹.

In a sixth aspect of the first embodiment:

each R^(17a) is selected from hydrogen, —CO₂H, and —CH₂CO₂H;

X′ is a chain of between 8 and 48 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two, orthree C(O)NH groups embedded therein; and wherein the chain isoptionally substituted with one or two groups independently selectedfrom —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and —CH₂CO₂H; provided that X′ isother than unsubstituted PEG; and

R³⁰ is selected from —CH₃, —CO₂H, and —C(O)NH₂.

In a seventh aspect of the first embodiment the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein:

A is

m and w are 1;

R¹⁴, R¹⁵, and R^(16a) are each hydrogen; and

R¹⁶ is —C(R^(17a))₂[C(O)N(R^(16a))C(R^(17a))₂]_(w′)—X—R³¹.

In an eighth aspect of the first embodiment:

each R^(17a) is selected from hydrogen, —CO₂H, and —CH₂CO₂H;

X is a chain of between 8 and 48 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two, orthree C(O)NH groups embedded therein; and wherein the chain isoptionally substituted with one or two groups independently selectedfrom —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and —CH₂CO₂H; and

R³¹ is selected from —CH₃, —CO₂H, and —C(O)NH₂.

In a ninth aspect of the first embodiment the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein:

A is

m and w are 1;

R¹⁴, R¹⁵, and R^(16a) are each hydrogen; and

R¹⁶ is —(C(R^(17a))(R¹⁷)C(O)NR^(16a))_(n′)—H.

In a tenth aspect of the first embodiment:

each R^(17a) is hydrogen; and

each R¹⁷ is selected from hydrogen, —CH₃, (CH₂)_(z)N₃, —(CH₂)_(z)NH₂,—X—R³¹, —(CH₂)_(z)CO₂H, —CH₂OH, CH₂C═CH, and —(CH₂)_(z)-triazolyl-X—R³⁵;provided at least one R¹⁷ is other than hydrogen, —CH₃, or —CH₂OH;

z is 1-4;

R³¹ is selected from —CH₃, —CO₂H, and —C(O)NH₂;

X is a chain of between 7 and 155 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two, orthree C(O)NH groups embedded therein; and wherein the chain isoptionally substituted with one or two groups independently selectedfrom —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and —CH₂CO₂H; and

R³⁵ is selected from —CO₂H, —C(O)NR^(w)R^(x), CH₃, biotin,-2-fluoropyridine, —C(O)—(CH₂)₂—C(O)O-vitamin E, and —C(O)O-vitamin E.

In an eleventh aspect of the first embodiment the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein:

A is

m and w are 1;

R¹⁴, R¹⁵, and R^(16a) are each hydrogen; and

R¹⁶ is —(CR^(17a))(R¹⁷)C(O)NR^(16a))_(m′)—C(R^(17a))(R¹⁷)—CO₂H.

In a twelfth aspect of the first embodiment:

m′ is 1-3;

each R^(17a) is hydrogen;

each R¹⁷ is selected from hydrogen, —CH₃, (CH₂)_(z)N₃, —(CH₂)_(z)NH₂,—X—R³¹, —(CH₂)_(z)CO₂H, —CH₂OH, CH₂C═CH, —(CH₂)_(z)-triazolyl-X—R³⁵, andC(O)O-vitamin E; and

provided at least one R¹⁷ is other than hydrogen, —CH₃, or —CH₂OH;

z is 1-4;

R³¹ is selected from —CH₃, —CO₂H, and —C(O)NH₂;

X is a chain of between 20 and 60 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two, orthree C(O)NH groups embedded therein; and wherein the chain isoptionally substituted with one or two groups independently selectedfrom —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and —CH₂CO₂H; and

R³⁵ is selected from —CO₂H, —C(O)NR^(w)R^(x), CH₃, biotin,2-fluoropyridine, —C(O)—(CH₂)₂—C(O)O-vitamin E, and —C(O)O-vitamin E.

In a thirteenth aspect of the first embodiment the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein R¹ is phenylC₁-C₃alkyl wherein the phenyl part isoptionally substituted with hydroxyl, halo, or methoxy; R² is C₁-C₇alkylor, R² and R^(b), together with the atoms to which they are attached,form a piperidine ring; R³ is NR^(x)R^(y)(C₁-C₇alkyl),NR^(u)R^(v)carbonylC₁-C₃alkyl, or carboxyC₁-C₃alkyl; R⁴ and R^(d),together with the atoms to which they are attached, form a pyrrolidinering; R⁵ is hydroxyC₁-C₃alkyl, imidazolylC₁-C₃alkyl, orNR^(x)R^(y)(C₁-C₇alkyl); R⁶ is carboxyC₁-C₃alkyl,NR^(u)R^(v)carbonylC₁-C₃alkyl, NR^(x)R^(y)(C₁-C₇alkyl), or C₁-C₇alkyl;R⁷ and R^(g), together with the atoms to which they are attached, form apyrrolidine ring optionally substituted with hydroxy; R⁸ and R¹⁰ arebenzothienyl or indolylC₁-C₃alkyl optionally substituted withcarboxyC₁-C₃alkyl; R⁹ is hydroxyC₁-C₃alkyl, aminoC₁-C₃alkyl, orC₁-C₇alkyl, R¹¹ is C₁-C₃alkoxyC₁-C₃alkyl or C₁-C₇alkyl; R¹² isC₁-C₇alkyl or hydroxyC₁-C₃alkyl; and R¹³ is C₁-C₇ alkyl,carboxyC₁-C₃alkyl, or —(CH₂)₃NHC(NH)NH₂.

In a fourteenth aspect of the first embodiment the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein:

A is

m and w are 1;

R¹⁴, R¹⁵, and R^(16a) are each hydrogen;

R^(d) is methyl or, R^(d) and R⁴, together with the atoms to which theyare attached, form a ring selected from azetidine, pyrolidine,morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein eachring is optionally substituted with one or two groups independentlyselected from amino, cyano, methyl, halo, hydroxy, and phenyl;

R^(g) is methyl or, R^(g) and R⁷, together with the atoms to which theyare attached,

form a ring selected from azetidine, pyrolidine, morpholine, piperidine,piperazine, and tetrahydrothiazole; wherein each ring is optionallysubstituted with one or two groups independently selected from amino,benzyl optionally substituted with a halo group, benzyloxy, cyano,cyclohexyl, methyl, halo, hydroxy, isoquinolinyloxy optionallysubstituted with a methoxy group, quinolinyloxy optionally substitutedwith a halo group, and tetrazolyl; and wherein the pyrrolidine and thepiperidine ring are optionally fused to a cyclohexyl, phenyl, or indolegroup; and

R^(k) is methyl or, R^(k) and R¹¹, together with the atoms to which theyare attached, form a ring selected from azetidine, pyrolidine,morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein eachring is optionally substituted with one or two groups independentlyselected from amino, cyano, methyl, halo, and hydroxy.

In a second embodiment the present disclosure provides a compound offormula (II)

or a pharmaceutically acceptable salt thereof, wherein:

A is selected from

wherein:

-   -   n is 0 or 1;

R¹⁴ and R¹⁵ are independently selected from hydrogen and methyl;

R^(16a) is selected from hydrogen and C₁-C₆ alkyl;

R¹⁶ is selected from

—(C(R^(17a))₂)₂—X—R³⁰,

—C(R^(17a))₂C(O)N(R^(16a))C(R^(17a))₂—X′—R³¹,

—C(R^(17a))₂[C(O)N(R^(16a))C(R^(17a))₂]_(w′)—X—R³¹,

—(C(R^(17a))(R¹⁷)C(O)NR^(16a))_(n′)—H; and

—(CR^(17a))(R¹⁷)C(O)NR^(16a))_(m′)—C(R^(17a))(R¹⁷)—CO₂H; wherein:

w′ is 2 or 3;

n′ is 1-6;

m′ is 1-5;

X is a chain of between 1 and 172 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two,three, or four groups selected from —NHC(O)NH—, and —C(O)NH embeddedtherein; and wherein the chain is optionally substituted with one to sixgroups independently selected from —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and—CH₂CO₂H,

X′ is a chain of between 1 and 172 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two,three, or four groups selected from —NHC(O)NH—, and —C(O)NH embeddedtherein; and wherein the chain is optionally substituted with one to sixgroups independently selected from —CO₂H, —C(O)NH₂, and —CH₂CO₂H,provided that X′ is other than unsubstituted PEG;

R³⁰ is selected from —CO₂H, —C(O)NR^(w)R^(x), and —CH₃ wherein R^(w) andR^(x) are independently selected from hydrogen and C₁-C₆alkyl, providedthat when X is all carbon, R³⁰ is other than —CH₃;

R³¹ is —CO₂H, —C(O)NR^(w)R^(x), —CH₃, alexa-5-SDP, and biotin;

each R^(17a) is independently selected from hydrogen, C₁-C₆alkyl,—CH₂OH, —CH₂CO₂H, —(CH₂)₂CO₂H,

each R¹⁷ is independently selected from hydrogen, —CH₃, (CH₂)_(z)N₃,—(CH₂)_(z)NH₂, —X—R³¹, —(CH₂)_(z)CO₂H, —CH₂OH, CH₂C═CH, and—(CH₂)_(z)-triazolyl-X—R³⁵, wherein z is 1-6 and R³⁵ is selected from—CO₂H, —C(O)NR^(w)R^(x), CH₃, biotin, -2-fluoropyridine,—C(O)—(CH₂)₂—C(O)O-vitamin E, —C(O)O-vitamin E, and

provided at least one R¹⁷ is other than hydrogen, —CH₃, or —CH₂OH;

R^(a), R^(f), R^(j), R^(k), R^(l), and R^(m) are hydrogen;

R^(b) and R^(c) are methyl;

R^(g) is selected from hydrogen and methyl;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹² are independentlyselected from a natural amino acid side chain and an unnatural aminoacid side chain or form a ring with the corresponding vicinal R group asdescribed below;

R^(d) is selected from hydrogen and methyl, or, R^(d) and R⁴, togetherwith the atoms to which they are attached, form a ring selected fromazetidine, pyrolidine, morpholine, piperidine, piperazine, andtetrahydrothiazole; wherein each ring is optionally substituted with oneto four groups independently selected from amino, cyano, methyl, halo,halomethyl, and hydroxy;

R^(e) is selected from hydrogen and methyl, or, R^(e) and R⁵, togetherwith the atoms to which they are attached, form a ring selected fromazetidine, pyrolidine, morpholine, piperidine, piperazine, andtetrahydrothiazole; wherein each ring is optionally substituted with oneto four groups independently selected from amino, cyano, methyl, halo,halomethyl, and hydroxy;

R^(h) is selected from hydrogen and methyl, or, R^(h) and R⁸, togetherwith the atoms to which they are attached, form a ring selected fromazetidine, pyrolidine, morpholine, piperidine, piperazine, andtetrahydrothiazole; wherein each ring is optionally substituted with oneto four groups independently selected from amino, cyano, methyl, halo,halomethyl, and hydroxy; and

R^(i) is selected from hydrogen and methyl, or, R^(i) and R⁹, togetherwith the atoms to which they are attached selected from azetidine,pyrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole;wherein each ring is optionally substituted with one to four groupsindependently selected from amino, cyano, methyl, halo, halomethyl, andhydroxy.

In a first aspect of the second embodiment the present disclosureprovides a compound of formula (II), or a pharmaceutically acceptablesalt thereof, wherein

A is

n is 1;

R¹⁶ is —(CR^(17a))(R¹⁷)C(O)NR^(16a))_(m′)—C(R^(17a))(R¹⁷)—CO₂H;

each R^(16a) is hydrogen;

m′ is 2, 3, or 4;

each R^(17a) is hydrogen;

each R¹⁷ is independently selected from hydrogen, —(CH₂)_(z)NH₂, —X—R³¹and —CH₂C═CH,

z is 4;

X is a chain of between 26 and 155 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two, orthree C(O)NH groups embedded therein; and wherein the chain isoptionally substituted with one or two groups independently selectedfrom —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and —CH₂CO₂H; and

R³¹ is —CH₃, alexa-5-SDP, and biotin.

In a third embodiment the present disclosure provides a method ofenhancing, stimulating, and/or increasing the immune response in asubject in need thereof, said method comprising administering to thesubject a therapeutically effective amount of a compound of formula (I)or a therapeutically acceptable salt thereof. In a first aspect of thethird embodiment the method further comprises administering anadditional agent prior to, after, or simultaneously with the compound offormula (I) or a therapeutically acceptable salt thereof. In a secondaspect the additional agent is an antimicrobial agent, an antiviralagent, a cytotoxic agent, and/or an immune response modifier.

In a fourth embodiment the present disclosure provides a method ofinhibiting growth, proliferation, or metastasis of cancer cells in asubject in need thereof, said method comprising administering to thesubject a therapeutically effective amount a compound of formula (I), ora therapeutically acceptable salt thereof. In a first aspect of thefourth embodiment the cancer is selected from melanoma, renal cellcarcinoma, squamous non-small cell lung cancer (NSCLC), non-squamousNSCLC, colorectal cancer, castration-resistant prostate cancer, ovariancancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma,squamous cell carcinoma of the head and neck, carcinomas of theesophagus, gastrointestinal tract and breast, and hematologicalmalignancies.

In a fifth embodiment the present disclosure provides a method oftreating an infectious disease in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a compound of formula (I) or a therapeutically acceptable saltthereof. In a first aspect of the fifth embodiment the infectiousdisease is caused by a virus. In a second aspect the virus is selectedfrom HIV, Hepatitis A, Hepatitis B, Hepatitis C, herpes viruses, andinfluenza.

In a sixth embodiment the present disclosure provides a method oftreating septic shock in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a compound of formula (I) or a therapeutically acceptable saltthereof.

In a seventh embodiment the present disclosure provides a method ofenhancing, stimulating, and/or increasing the immune response in asubject in need thereof, said method comprising administering to thesubject a therapeutically effective amount of a compound of formula (II)or a therapeutically acceptable salt thereof. In a first aspect of theseventh embodiment the method further comprises administering anadditional agent prior to, after, or simultaneously with the compound offormula (II) or a therapeutically acceptable salt thereof. In a secondaspect the additional agent is an antimicrobial agent, an antiviralagent, a cytotoxic agent, and/or an immune response modifier. In a thirdaspect the additional agent is an HDAC inhibitor. In a fourth embodimentthe additional agent is a TLR7 and/or TLR8 agonist.

In an eighth embodiment the present disclosure provides a method ofinhibiting growth, proliferation, or metastasis of cancer cells in asubject in need thereof, said method comprising administering to thesubject a therapeutically effective amount a compound of formula (II),or a therapeutically acceptable salt thereof. In a first aspect of theeighth embodiment the cancer is selected from melanoma, renal cellcarcinoma, squamous non-small cell lung cancer (NSCLC), non-squamousNSCLC, colorectal cancer, castration-resistant prostate cancer, ovariancancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma,squamous cell carcinoma of the head and neck, carcinomas of theesophagus, gastrointestinal tract and breast, and hematologicalmalignancies.

In a ninth embodiment the present disclosure provides a method oftreating an infectious disease in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a compound of formula (II) or a therapeutically acceptablesalt thereof. In a first aspect of the ninth embodiment the infectiousdisease is caused by a virus. In a second aspect the virus is selectedfrom HIV, Hepatitis A, Hepatitis B, Hepatitis C, herpes viruses, andinfluenza.

In a tenth embodiment the present disclosure provides a method oftreating septic shock in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a compound of formula (II) or a therapeutically acceptablesalt thereof.

In another embodiment the present disclosure provides a compound offormula (III)

or a pharmaceutically acceptable salt thereof, wherein:

A is selected from

wherein:

denotes the point of attachment to the carbonyl group and

denotes the point of attachment to the nitrogen atom;

n is 0 or 1;

m is 1 or 2;

w is 0, 1, or 2;

R¹⁴ and R¹⁵ are independently selected from hydrogen and methyl;

R^(16a) is selected from hydrogen and C₁-C₆ alkyl;

R¹⁶ is selected from

—(C(R^(17a))₂)₂—X—R³⁰,

—C(R^(17a))₂C(O)N(R^(16a))C(R^(17a))₂—X′—R³¹,

—C(R^(17a))₂[C(O)N(R^(16a))C(R^(17a))₂]_(w′)—X—R³¹,

—(C(R^(17a))(R¹⁷)C(O)NR^(16a))_(n′)—H; and

—(C(R^(17a))(R¹⁷)C(O)NR^(16a))_(m′)—C(R^(17a))(R¹⁷)—CO₂H;

wherein:

w′ is 2 or 3;

n′ is 1-6; (2,3)

m′ is 0-5; (1, 2, 3)

X is a chain of between 1 and 172 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two,three, or four groups selected from —NHC(O)NH—, and —C(O)NH— embeddedtherein; and wherein the chain is optionally substituted with one to sixgroups independently selected from —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and—CH₂CO₂H,

X′ is a chain of between 1 and 172 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two,three, or four groups selected from —NHC(O)NH—, and —C(O)NH— embeddedtherein; and wherein the chain is optionally substituted with one to sixgroups independently selected from —CO₂H, —C(O)NH₂, and —CH₂CO₂H,provided that X′ is other than unsubstituted PEG;

R³⁰ is selected from —CO₂H, —C(O)NR^(w)R^(x), and —CH₃ wherein R^(w) andR^(x) are independently selected from hydrogen and C₁-C₆alkyl, providedthat when X is all carbon, R³⁰ is other than —CH₃;

R³¹ is —CO₂H, —C(O)NR^(w)R^(x), —CH₃, alexa-5-SDP, and biotin;

each R^(17a) is independently selected from hydrogen, C₁-C₆alkyl,—CH₂OH, —CH₂CO₂H, —(CH₂)₂CO₂H,

each R¹⁷ is independently selected from hydrogen, —CH₃, (CH₂)_(z)N₃,—(CH₂)_(z)NH₂, —(CH₂)_(z)CO₂H, —CH₂OH, CH₂C═CH, and—(CH₂)_(z)-triazolyl-X—R³⁵, wherein z is 1-6 and R³⁵ is selected from—CO₂H, —C(O)NR^(w)R^(x), CH₃, biotin, -2-fluoropyridine,—C(O)—(CH₂)₂—C(O)O-vitamin E, and —C(O)O-vitamin E; provided at leastone R¹⁷ is other than hydrogen, —CH₃, or —CH₂OH;

R^(c), R^(f), R^(h), R^(i), R^(m), and R^(n) are hydrogen;

R^(a), R^(e), R^(j), and R^(k), are each independently selected fromhydrogen and methyl;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³ areindependently selected from a natural amino acid side chain and anunnatural amino acid side chain or form a ring with the correspondingvicinal R group as described below;

R^(e) and R^(k) can each form a ring with the corresponding vicinal Rgroup and the atoms to which they are attached selected from azetidine,pyrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole;wherein each ring is optionally substituted with one to four groupsindependently selected from amino, cyano, methyl, halo, and hydroxy;

R^(b) is methyl or, R^(b) and R², together with the atoms to which theyare attached, form a ring selected from azetidine, pyrolidine,morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein eachring is optionally substituted with one to four groups independentlyselected from amino, cyano, methyl, halo, and hydroxy;

R^(d) is hydrogen or methyl, or, R^(d) and R⁴, together with the atomsto which they are attached, can form a ring selected from azetidine,pyrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole;wherein each ring is optionally substituted with one to four groupsindependently selected from amino, cyano, methyl, halo, hydroxy, andphenyl;

R^(g) is hydrogen or methyl or R^(g) and R⁷, together with the atoms towhich they are attached, can form a ring selected from azetidine,pyrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole;wherein each ring is optionally substituted with one to four groupsindependently selected from amino, benzyl optionally substituted with ahalo group, benzyloxy, cyano, cyclohexyl, methyl, halo, hydroxy,isoquinolinyloxy optionally substituted with a methoxy group,quinolinyloxy optionally substituted with a halo group, and tetrazolyl;and wherein the pyrrolidine and the piperidine ring are optionally fusedto a cyclohexyl, phenyl, or indole group; and

R¹ is methyl or, R¹ and R¹², together with the atoms to which they areattached, form a ring selected from azetidine and pyrolidine, whereineach ring is optionally substituted with one to four independentlyselected from amino, cyano, methyl, halo, and hydroxy.

In another embodiment the present disclosure provides a compound offormula (IV)

or a pharmaceutically acceptable salt thereof, wherein:

A is selected from

wherein:

n is 0 or 1;

R¹⁴ and R¹⁵ are independently selected from hydrogen and methyl;

R^(16a) is selected from hydrogen and C₁-C₆ alkyl;

R¹⁶ is selected from

—(C(R^(17a))₂)₂—X—R³⁰,

—C(R^(17a))₂C(O)N(R^(16a))C(R^(17a))₂—X′—R³¹,

—C(R^(17a))₂[C(O)N(R^(16a))C(R^(17a))₂]_(w′)—X—R³¹,

—(C(R^(17a))(R¹⁷)C(O)NR^(16a))_(n′)—H; and

—(CR^(17a))(R¹⁷)C(O)NR^(16a))_(m′)—(R^(17a))(R¹⁷)—CO₂H; wherein:

w′ is 2 or 3;

n′ is 1-6;

m′ is 1-5;

X is a chain of between 1 and 172 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two,three, or four groups selected from —NHC(O)NH—, and —C(O)NH embeddedtherein; and wherein the chain is optionally substituted with one to sixgroups independently selected from —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and—CH₂CO₂H,

X′ is a chain of between 1 and 172 atoms wherein the atoms are selectedfrom carbon and oxygen and wherein the chain may contain one, two,three, or four groups selected from —NHC(O)NH—, and —C(O)NH embeddedtherein; and wherein the chain is optionally substituted with one to sixgroups independently selected from —CO₂H, —C(O)NH₂, and —CH₂CO₂H,provided that X′ is other than unsubstituted PEG;

R³⁰ is selected from —CO₂H, —C(O)NR^(w)R^(x), and —CH₃ wherein R^(w) andR^(x) are independently selected from hydrogen and C₁-C₆alkyl, providedthat when X is all carbon, R³⁰ is other than —CH₃;

R³¹ is —CO₂H, —C(O)NR^(w)R^(x), —CH₃, alexa-5-SDP, and biotin;

each R^(17a) is independently selected from hydrogen, C₁-C₆alkyl,—CH₂OH, —CH₂CO₂H, —(CH₂)₂CO₂H,

each R¹⁷ is independently selected from hydrogen, —CH₃, (CH₂)_(z)N₃,—(CH₂)_(z)NH₂, —X—R³¹, —(CH₂)_(z)CO₂H, —CH₂OH, CH₂C═CH, and—(CH₂)_(z)-triazolyl-X—R³⁵, wherein z is 1-6 and R³⁵ is selected from—CO₂H, —C(O)NR^(w)R^(x), CH₃, biotin, -2-fluoropyridine,—C(O)—(CH₂)₂—C(O)O-vitamin E, and —C(O)O-vitamin E; provided at leastone R¹⁷ is other than hydrogen, —CH₃, or —CH₂OH;

R^(a), R^(f), R^(j), R^(k), R^(l), and R^(m) are hydrogen;

R^(b) and R^(c) are methyl;

R^(g) is selected from hydrogen and methyl;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² are independentlyselected from a natural amino acid side chain and an unnatural aminoacid side chain or form a ring with the corresponding vicinal R group asdescribed below;

R^(d) is selected from hydrogen and methyl, or, R^(d) and R⁴, togetherwith the atoms to which they are attached, form a ring selected fromazetidine, pyrolidine, morpholine, piperidine, piperazine, andtetrahydrothiazole; wherein each ring is optionally substituted with oneto four groups independently selected from amino, cyano, methyl, halo,halomethyl, and hydroxy;

R^(e) is selected from hydrogen and methyl, or, R^(e) and R⁵, togetherwith the atoms to which they are attached, form a ring selected fromazetidine, pyrolidine, morpholine, piperidine, piperazine, andtetrahydrothiazole; wherein each ring is optionally substituted with oneto four groups independently selected from amino, cyano, methyl, halo,halomethyl, and hydroxy;

R^(h) is selected from hydrogen and methyl, or, R^(h) and R⁸, togetherwith the atoms to which they are attached, form a ring selected fromazetidine, pyrolidine, morpholine, piperidine, piperazine, andtetrahydrothiazole; wherein each ring is optionally substituted with oneto four groups independently selected from amino, cyano, methyl, halo,halomethyl, and hydroxy; and

R^(i) is selected from hydrogen and methyl, or, R^(i) and R⁹, togetherwith the atoms to which they are attached selected from azetidine,pyrolidine, morpholine, piperidine, piperazine, and tetrahydrothiazole;wherein each ring is optionally substituted with one to four groupsindependently selected from amino, cyano, methyl, halo, halomethyl, andhydroxy.

In compounds of formula (I) and (II) where the R side chains are part ofa ring that is substituted with methyl, it is understood that the methylgroup may be on any substitutable carbon atom in the ring, including thecarbon that is part of the macrocyclic parent structure.

The following groups are preferred at each R position. The amino acidsmay be D- or L-stereochemistry and may be substituted as describedelsewhere in the disclosure.

In compounds of formula (I), preferred R¹ side chains are:phenylalanine, tyrosine, 3-thien-2-yl, 4-methylphenylalanine,4-chlorophenylalanine, 3-methoxyphenylalanine, isotryptophan,3-methylphenylalanine, 1-naphthylalanine, 3,4-difluorophenylalanine,4-fluorophenylalanine, 3,4-dimethoxyphenylalanine,3,4-dichlorophenylalanine, 4-difluoromethylphenylalanine,2-methylphenylalanine, 2-naphthylalanine, tryptophan, 4-pyridinyl,4-bromophenylalanine, 3-pyridinyl, 4-trifluoromethylphenylalanine,4-carboxyphenylalanine, 4-methoxyphenylalanine, biphenylalanine, and3-chlorophenylalanine; and 2,4-diaminobutane.

In compounds of formula (I) where R² is not part of a ring, preferred R²side chains are: alanine, serine, and glycine.

In compounds of formula (I), preferred R³ side chains are: asparagine,aspartic acid, glutamic acid, glutamine, serine, ornithine, lysine,histidine, threonine, leucine, alanine, 2,3-diaminopropane, and2,4-diaminobutane.

In compounds of formula (I) where R⁴ is not part of a ring, preferred R⁴side chains are: valine, alanine, isoleucine, and glycine.

In compounds of formula (I), preferred R⁵ side chains are: aminomethane,histidine, asparagine, 2,3-diaminopropane, serine, glycine,2,4-diaminobutane, threonine, alanine, lysine, aspartic acid, alanine,and 3-thiazolylalanine.

In compounds of formula (I), preferred R⁶ side chains are: leucine,aspartic acid, asparagine, glutamic acid, glutamine, serine, lysine,3-cyclohexane, threonine, ornithine, 2,4-diaminobutane, alanine,arginine, and ornithine (COCH₃).

In compounds of formula (I) where R⁷ is not part of a ring, preferred R⁷side chains are: glycine, 2,4-diaminobutane, serine, lysine, arginine,ornithine, histidine, asparagine, glutamine, alanine, and2,4-diaminobutane (C(O)cyclobutane).

In compounds of formula (I) preferred R⁸ side chains are tryptophan and1,2-benzisothiazolinylalanine.

In compounds of formula (I) preferred R⁹ side chains are: serine,histidine, lysine, ornithine, 2,4-dibutylamine, threonine, lysine,glycine, glutamic acid, valine, 2,3-diaminopropane, arginine, asparticacid, and tyrosine.

In compounds of formula (I) preferred R¹⁰ side chains are: optionallysubstituted tryptophan, benzisothiazolylalanine, 1-naphthylalanine,methionine.

In compounds of formula (I) preferred R¹¹ side chains are: norleucine,leucine, asparagine, phenylalanine, methionine, ethoxymethane, alanine,tryptophan, isoleucine, phenylpropane, glutamic acid, hexane, andheptane.

In compounds of formula (I) where R¹² is not part of a ring, preferredR¹² side chains are: norleucine, alanine, ethoxymethane, methionine,serine, phenylalanine, methoxyethane, leucine, tryptophan, isoleucine,glutamic acid, hexane, heptane, and glycine.

In compounds of formula (I) preferred R¹³ side chains: arginine,ornithine, alanine, 2,4-diaminobutane, 2,3-diaminopropane, leucine,aspartic acid, glutamic acid, serine, lysine, threonine,cyclopropylmethane, glycine, valine, isoleucine, histidine, and2-aminobutane.

In accordance with the present disclosure, we have discovered peptidesthat specifically bind to PD-L1 and are capable of inhibiting theinteraction of PD-L1 with PD-1 and CD80. These macrocyclic peptidesexhibit in vitro immunomodulatory efficacy thus making them therapeuticcandidates for the treatment of various diseases including cancer andinfectious diseases.

The terms “specific binding” or “specifically bind” refer to theinteraction between a protein and a binding molecule, such as a compoundor ligand. The interaction is dependent upon the presence of aparticular structure (i.e., an enzyme binding site, an antigenicdeterminant or epitope) of the protein that is recognized by the bindingmolecule. For example, if a compound has specific binding for proteinbinding site “A”, the presence of the compound in a reaction containinga protein including binding site A, and a labeled peptide thatspecifically binds to protein binding site A will reduce the amount oflabeled peptide bound to the protein. In contrast, nonspecific bindingof a compound to the protein does not result in aconcentration-dependent displacement of the labeled peptide from theprotein.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include deuteriumand tritium. Isotopes of carbon include ¹³C and ¹⁴C.Isotopically-labeled compounds of the invention can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described herein, using an appropriateisotopically-labeled reagent in place of the non-labeled reagentotherwise employed. Such compounds may have a variety of potential uses,for example as standards and reagents in determining biologicalactivity. In the case of stable isotopes, such compounds may have thepotential to favorably modify biological, pharmacological, orpharmacokinetic properties.

An additional aspect of the subject matter described herein is the useof the disclosed peptides as radiolabeled ligands for development ofligand binding assays or for monitoring of in vivo adsorption,metabolism, distribution, receptor binding or occupancy, or compounddisposition. For example, a macrocyclic peptide described herein may beprepared using the radioactive isotope ¹²⁵I and the resultingradiolabeled peptide may be used to develop a binding assay or formetabolism studies. Alternatively, and for the same purpose, amacrocyclic peptide described herein may be converted to a radiolabeledform by catalytic tritiation using methods known to those skilled in theart.

The macrocyclic peptides of the present disclosure can also be used asPET imaging agents by adding a radioactive tracer using methods known tothose skilled in the art.

Preferred peptides include at least one of the macrocyclic peptidesprovided herein and these peptides may be included in pharmaceuticalcompositions and combinations.

The definitions provided herein apply, without limitation, to the termsas used throughout this specification, unless otherwise limited inspecific instances.

Those of ordinary skill in the art of amino acid and peptide chemistryare aware that an amino acid includes a compound represented by thegeneral structure:

where R and R′ are as discussed herein.

Unless otherwise indicated, the term “amino acid” as employed herein,alone or as part of another group, includes, without limitation, anamino group and a carboxyl group linked to the same carbon, referred toas “α” carbon, where R and/or R′ can be a natural or an un-natural sidechain, including hydrogen. The absolute “S” configuration at the “α”carbon is commonly referred to as the “L” or “natural” configuration. Inthe case where both the “R” and the “R′” (prime) substituents equalhydrogen, the amino acid is glycine and is not chiral.

The terms “natural amino acid side chain” and “naturally occurring aminoacid side chain,” as used herein, refer to side chain of any of thenaturally occurring amino acids (i.e., alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine,-histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine) usually in theS-configuration (i.e., the L-amino acid).

The terms “unnatural amino acid side chain” and “non-naturally occurringamino acid side chain,” as used herein, refer to a side chain of anynaturally occurring amino acid usually in the R-configuration (i.e., theD-amino acid) or to a group other than a naturally occurring amino acidside chain in R- or S-configuration (i.e., the D- or L-amino acid,respectively) selected from:

C₂-C₇alkenyl, C₁-C₃alkoxyC₁-C₃alkyl, C₁-C₆alkoxycarbonylC₁-C₃alkyl,C₁-C₇alkyl, C₁-C₃alkylsulfanylC₁-C₃alkyl, amidoC₁-C₃alkyl,aminoC₁-C₃alkyl, azaindolylC₁-C₃alkyl, benzothiazolylC₁-C₃alkyl,benzothienylC₁-C₃alkyl, benzyloxyC₁-C₃alkyl, carboxyC₁-C₃alkyl,C₃-C₁₄cycloalkylC₁-C₃alkyl, diphenylmethyl, furanylC₁-C₃alkyl,imidazolylC₁-C₃alkyl, naphthylC₁-C₃alkyl, pyridinylC₁-C₃alkyl,thiazolylC₁-C₃alkyl, thienylC₁-C₃alkyl;

biphenylC₁-C₃alkyl wherein the biphenyl is optionally substituted with amethyl group;

heterocyclyl optionally substituted with one, two, three, four, or fivegroups independently selected from C₁-C₄alkoxy, C₁-C₄alkyl,C₁-C₃alkylsulfonylamino, amido, amino, aminoC₁-C₃alkyl, aminosulfonyl,carboxy, cyano, halo, haloC₁-C₃alkyl, hydroxy, —NC(NH₂)₂, nitro, and—OP(O)(OH)₂;

indolylC₁-C₃alkyl, wherein the indolyl part is optionally substitutedwith one group selected from C₁-C₃alkyl, carboxyC₁-C₃alkyl, halo,hydroxy, and phenyl, wherein the phenyl is further optionallysubstituted by one, two, or three groups independently selected fromC₁-C₃alkoxy, C₁-C₃alkyl, and halo;

NR^(x)R^(y)(C₁-C₇alkyl), wherein R^(x) and R^(y) are independentlyselected from hydrogen, C₂-C₄alkenyloxycarbonyl, C₁-C₃alkyl,C₁-C₃alkylcarbonyl, C₃-C₁₄cycloalkylcarbonyl, furanylcarbonyl, andphenylcarbonyl. When the alkyl linker contains more than one carbon anadditional NR^(x)R^(y) group can be on the chain.

NR^(u)R^(v)carbonylC₁-C₃alkyl, wherein R^(u) and R^(v) are independentlyselected from hydrogen, C₁-C₃alkyl, and triphenylmethyl;

phenyl optionally substituted with one, two, three, four, or five groupsindependently selected from C₁-C₄alkoxy, C₁-C₄alkyl,C₁-C₃alkylsulfonylamino, amido, amino, aminoC₁-C₃alkyl, aminosulfonyl,carboxy, cyano, halo, haloC₁-C₃alkyl, hydroxy, —NC(NH₂)₂, nitro, and—OP(O)(OH)₂;

phenylC₁-C₃alkyl wherein the phenyl part is optionally substituted withone, two, three, four, or five groups independently selected fromC₁-C₄alkoxy, C₁-C₄alkyl, C₁-C₃alkylsulfonylamino, amido, amino,aminoC₁-C₃alkyl, aminosulfonyl, carboxy, cyano, halo, haloC₁-C₃alkyl,hydroxy, —NC(NH₂)₂, nitro, and —OP(O)(OH)₂; and

phenoxyC₁-C₃alkyl wherein the phenyl is optionally substituted with aC₁-C₃alkyl group.

The term “alexa-5-SDP,” as used herein, refers to

wherein W is O or NH.

The term “C₂-C₄alkenyl,” as used herein, refers to a straight orbranched chain group of two to four carbon atoms containing at least onecarbon-carbon double bond.

The term “C₂-C₇alkenyl,” as used herein, refers to a straight orbranched chain group of two to seven carbon atoms containing at leastone carbon-carbon double bond.

The term “C₂-C₄alkenyloxy,” as used herein, refers to a C₂-C₄alkenylgroup attached to the parent molecular moiety through an oxygen atom.

The term “C₁-C₃alkoxy,” as used herein, refers to a C₁-C₃alkyl groupattached to the parent molecular moiety through an oxygen atom.

The term “C₁-C₄alkoxy,” as used herein, refers to a C₁-C₄alkyl groupattached to the parent molecular moiety through an oxygen atom.

The term “C₁-C₆alkoxy,” as used herein, refers to a C₁-C₆alkyl groupattached to the parent molecular moiety through an oxygen atom.

The term “C₁-C₃alkoxyC₁-C₃alkyl,” as used herein, refers to aC₁-C₃alkoxy group attached to the parent molecular moiety through aC₁-C₃alkyl group. The term “C₁-C₆alkoxycarbonyl,” as used herein, refersto a C₁-C₆alkoxy group attached to the parent molecular moiety through acarbonyl group.

The term “C₁-C₆alkoxycarbonylC₁-C₃alkyl,” as used herein, refers to aC₁-C₆alkoxycarbonyl group attached to the parent molecular moietythrough a C₁-C₃alkyl group.

The term “C₁-C₃alkyl,” as used herein, refers to a group derived from astraight or branched chain saturated hydrocarbon containing from one tothree carbon atoms.

The term “C₁-C₄alkyl,” as used herein, refers to a group derived from astraight or branched chain saturated hydrocarbon containing from one tofour carbon atoms.

The term “C₁-C₆alkyl,” as used herein, refers to a group derived from astraight or branched chain saturated hydrocarbon containing from one tosix carbon atoms.

The term “C₁-C₃alkylcarbonyl,” as used herein, refers to a C₁-C₃alkylgroup attached to the parent molecular moiety through a carbonyl group.

The term “C₁-C₃alkylsulfanyl,” as used herein, refers to a C₁-C₃alkylgroup attached to the parent molecular moiety through a sulfur atom.

The term “C₁-C₃alkylsulfanylC₁-C₃alkyl,” as used herein, refers to aC₁-C₃alkylsulfanyl group attached to the parent molecular moiety througha C₁-C₃alkyl group.

The term “C₁-C₃alkylsulfonyl,” as used herein, refers to a C₁-C₃alkylgroup attached to the parent molecular moiety through a sulfonyl group.

The term “C₁-C₃alkylsulfonylamino,” as used herein, refers to aC₁-C₃alkylsulfonyl group attached to the parent molecular moiety throughan amino group.

The term “amido,” as used herein, refers to —C(O)NH₂.

The term “amidoC₁-C₃alkyl,” as used herein, refers to an amido groupattached to the parent molecular moiety through a C₁-C₃alkyl group.

The term “amino,” as used herein, refers to —NH₂.

The term “aminoC₁-C₃alkyl,” as used herein, refers to an amino groupattached to the parent molecular moiety through a C₁-C₃alkyl group.

The term “aminosulfonyl,” as used herein, refers to an amino groupattached to the parent molecular moiety through a sulfonyl group.

The term “azaindolylC₁-C₃alkyl,” as used herein, refers to an azaindolylgroup attached to the parent molecular through a C₁-C₃alkyl group. Theazaindolyl group can be attached to the alkyl moiety through anysubstitutable atom in the group.

The term “benzothiazolylC₁-C₃alkyl,” as used herein, refers to anbenzothiazolyl group attached to the parent molecular through aC₁-C₃alkyl group. The benzothiazolyl group can be attached to the alkylmoiety through any substitutable atom in the group.

The term “benzothienylC₁-C₃alkyl,” as used herein, refers to abenzothienyl group attached to the parent molecular through a C₁-C₃alkylgroup. The benzothienyl group can be attached to the alkyl moietythrough any substitutable atom in the group.

The term “benzyloxy,” as used herein, refers to a benzyl group attachedto the parent molecular moiety through an oxygen atom.

The term “benzyloxyC₁-C₃alkyl,” as used herein, refers to a benzyloxygroup attached to the parent molecular moiety through a C₁-C₃alkylgroup.

The term “biotin,” as used herein, refers to:

wherein W is O or NH.

The term “biphenylC₁-C₃alkyl,” as used herein, refers to a biphenylgroup attached to the parent molecular moiety through a C₁-C₃alkylgroup. The biphenyl group can be attached to the alkyl moiety throughany substitutable atom in the group.

The term “carbonyl,” as used herein, refers to —C(O)—.

The term “carboxy,” as used herein, refers to —CO₂H.

The term “carboxyC₁-C₃alkyl,” as used herein, refers to a carboxy groupattached to the parent molecular moiety through a C₁-C₃alkyl group.

The term “cyano,” as used herein, refers to —CN.

The term “C₃-C₁₄cycloalkyl,” as used herein, refers to a saturatedmonocyclic, bicyclic, or tricyclic hydrocarbon ring system having threeto fourteen carbon atoms and zero heteroatoms. The bicyclic andtricyclic rings may be fused, spirocyclic, or bridged. Representativeexamples of cycloalkyl groups include, but are not limited to,cyclopropyl, cyclopentyl, bicyclo[3.1.1]heptyl, and adamantyl.

The term “C₃-C₁₄cycloalkylC₁-C₃alkyl,” as used herein, refers to aC₃-C₁₄cycloalkyl group attached to the parent molecular moiety through aC₁-C₃alkyl group.

The term “C₃-C₁₄cycloalkylcarbonyl,” as used herein, refers to a C₃-C₁₄cycloalkyl group attached to the parent molecular moiety through acarbonyl group.

The term “furanylC₁-C₃alkyl,” as used herein, refers to a furanyl groupattached to the parent molecular moiety through a C₁-C₃alkyl group. Thefuranyl group can be attached to the alkyl moiety through anysubstitutable atom in the group.

The term “furanylcarbonyl,” as used herein, refers to a furanyl groupattached to the parent molecular moiety through a carbonyl group.

The terms “halo” and “halogen,” as used herein, refer to F, Cl, Br, orI.

The term “haloC₁-C₃alkyl,” as used herein, refers to a C₁-C₃alkyl groupsubstituted with one, two, or three halogen atoms.

The term “halomethyl,” as used herein, refers to a methyl groupsubstituted with one, two, or three halogen atoms.

The term “heterocyclyl,” as used herein, refers to a five-, six-, orseven-membered ring containing one, two, or three heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Thefive-membered ring has zero to two double bonds and the six- andseven-membered rings have zero to three double bonds. The term“heterocyclyl” also includes bicyclic groups in which the heterocyclylring is fused to a four- to six-membered aromatic or non-aromaticcarbocyclic ring or another monocyclic heterocyclyl group. Theheterocyclyl groups of the present disclosure are attached to the parentmolecular moiety through a carbon atom in the group. Examples ofheterocyclyl groups include, but are not limited to, benzothienyl,furyl, imidazolyl, indolinyl, indolyl, isothiazolyl, isoxazolyl,morpholinyl, oxazolyl, piperazinyl, piperidinyl, pyrazolyl, pyridinyl,pyrrolidinyl, pyrrolopyridinyl, pyrrolyl, thiazolyl, thienyl, andthiomorpholinyl.

The term “hydroxy,” as used herein, refers to —OH.

The term “imidazolylC₁-C₃alkyl,” as used herein, refers to an imidazolylgroup attached to the parent molecular moiety through a C₁-C₃alkylgroup. The imidazolyl group can be attached to the alkyl moiety throughany substitutable atom in the group.

The term “indolylC₁-C₃alkyl,” as used herein, refers to an indolyl groupattached to the parent molecular moiety through a C₁-C₃alkyl group. Theindolyl group can be attached to the alkyl moiety through anysubstitutable atom in the group.

The term “naphthylC₁-C₃alkyl,” as used herein, refers to a naphthylgroup attached to the parent molecular moiety through a C₁-C₃alkylgroup. The naphthyl group can be attached to the alkyl moiety throughany substitutable atom in the group.

The term “nitro,” as used herein, refers to —NO₂.

The term “NR^(x)R^(y),” as used herein, refers to two groups, R^(a) andR^(b), which are attached to the parent molecular moiety through anitrogen atom. R^(a) and R^(b) are independently selected from hydrogen,C₂-C₄alkenyloxycarbonyl, C₁-C₃alkylcarbonyl, C₃-C₁₄cycloalkylcarbonyl,furanylcarbonyl, and phenylcarbonyl.

The term “NR^(x)R^(y)(C₁-C₃)alkyl,” as used herein, refers to anNR^(x)R^(y) group attached to the parent molecular moiety through aC₁-C₃alkyl group.

The term “NR^(u)R^(v),” as used herein, refers to two groups, R^(u) andR^(v), which are attached to the parent molecular moiety through anitrogen atom. R^(u) and R^(v) are independently selected from hydrogen,C₁-C₃alkyl, and triphenylmethyl.

The term “NR^(u)R^(v)carbonyl,” as used herein, refers to an NR^(u)R^(v)group attached to the parent molecular moiety through a carbonyl group.

The term “NR^(u)R^(v)carbonylC₁-C₃alkyl,” as used herein, refers to anNR^(u)R^(v)carbonyl group attached to the parent molecular moietythrough a C₁-C₃alkyl group.

The term “PEG,” as used herein, refers to polyethylene glycol, a polymerof ethylene oxide represented by the formula

wherein n is between 1 and 57. It should be understood that the PEGgroup may be attached to the parent molecular moiety through the oxygenatom or the carbon atom.

The tem “phenoxy,” as used herein, refers to a phenyl group attached tothe parent molecular moiety through an oxygen atom.

The term “phenoxyC₁-C₃alkyl,” as used herein, refers to a phenoxy groupattached to the parent molecular moiety through a C₁-C₃alkyl group.

The term “phenylC₁-C₃alkyl,” as used herein, refers to a phenyl groupattached to the parent molecular moiety through a C₁-C₃alkyl group.

The term “phenylcarbonyl,” as used herein, refers to a phenyl groupattached to the parent molecular moiety through a carbonyl group.

The term “pyridinylC₁-C₃alkyl,” as used herein, refers to a pyridinylgroup attached to the parent molecular moiety through a C₁-C₃alkylgroup. The pyridinyl group can be attached to the alkyl moiety throughany substitutable atom in the group.

The term “sulfanyl,” as used herein, refers to —S—.

The term “sulfonyl,” as used herein, refers to —SO₂—.

The term “thiazolylC₁-C₃alkyl,” as used herein, refers to a thiazolylgroup attached to the parent molecular moiety through a C₁-C₃alkylgroup. The thiazolyl group can be attached to the alkyl moiety throughany substitutable atom in the group.

The term “thienylC₁-C₃alkyl,” as used herein, refers to a thienyl groupattached to the parent molecular moiety through a C₁-C₃alkyl group. Thethienyl group can be attached to the alkyl moiety through anysubstitutable atom in the group.

The term “treating” refers to: (i) preventing a disease, disorder, orcondition from occurring in a patient that may be predisposed to thedisease, disorder, and/or condition but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, disorder, or condition, i.e.,arresting its development; and (iii) relieving the disease, disorder, orcondition, i.e., causing regression of the disease, disorder, and/orcondition and/or symptoms associated with the disease, disorder, and/orcondition.

The term “vitamin E,” as used herein refers to:

Binding of the macrocyclic peptides to PD-L1 can be measured, forexample, by methods such as homogeneous time-resolved fluorescence(HTRF), Surface Plasmon Resonance (SPR), isothermal titrationcalorimetry (ITC), nuclear magnetic resonance spectroscopy (NMR), andthe like. Further, binding of the macrocyclic peptides to PD-L1expressed on the surface of cells can be measured as described herein incellular binding assays.

Administration of a therapeutic agent described herein includes, withoutlimitation, administration of a therapeutically effective amount oftherapeutic agent. The term “therapeutically effective amount” as usedherein refers, without limitation, to an amount of a therapeutic agentto treat or prevent a condition treatable by administration of acomposition of the PD-1/PD-L1 binding inhibitors described herein. Thatamount is the amount sufficient to exhibit a detectable therapeutic orpreventative or ameliorative effect. The effect may include, for exampleand without limitation, treatment or prevention of the conditions listedherein. The precise effective amount for a subject will depend upon thesubject's size and health, the nature and extent of the condition beingtreated, recommendations of the treating physician, and therapeutics orcombination of therapeutics selected for administration. Thus, it is notuseful to specify an exact effective amount in advance.

In another aspect, the disclosure pertains to methods of inhibitinggrowth of tumor cells in a subject using the macrocyclic peptides of thepresent disclosure. As demonstrated herein, the macrocyclic peptides ofthe present disclosure are capable of binding to PD-L1, disrupting theinteraction between PD-L1 and PD-1, competing with the binding of PD-L1with anti-PD-1 monoclonal antibodies that are known to block theinteraction with PD-1, enhancing CMV-specific T cell IFNγ secretion, andenhancement of HIV-specific T cell IFNg secretion. As a result, themacrocyclic peptides of the present disclosure are useful for modifyingan immune response, treating diseases such as cancer or infectiousdisease, stimulating a protective autoimmune response or to stimulateantigen-specific immune responses (e.g., by coadministration of PD-L1blocking peptides with an antigen of interest).

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “Programmed Death Ligand 1”, “Programmed Cell Death Ligand 1”,“Protein PD-L1”, “PD-L1”, “PDL1”, “PDCDL1”, “hPD-L1”, “hPD-LI”, “CD274”and “B7-H1” are used interchangeably, and include variants, isoforms,species homologs of human PD-L1, and analogs having at least one commonepitope with PD-L1. The complete PD-L1 sequence can be found underGENBANK® Accession No. NP_054862.

The terms “Programmed Death 1”, “Programmed Cell Death 1”, “ProteinPD-1”, “PD-1”, “PD1”, “PDCD1”, “hPD-1” and “hPD-I” are usedinterchangeably, and include variants, isoforms, species homologs ofhuman PD-1, and analogs having at least one common epitope with PD-1.The complete PD-1 sequence can be found under GENBANK® Accession No.U64863.

The terms “cytotoxic T lymphocyte-associated antigen-4”, “CTLA-4”,“CTLA4”, “CTLA-4 antigen” and “CD152” (see, e.g., Murata, Am. J.Pathol., 155:453-460 (1999)) are used interchangeably, and includevariants, isoforms, species homologs of human CTLA-4, and analogs havingat least one common epitope with CTLA-4 (see, e.g., Balzano, Int. J.Cancer Suppl., 7:28-32 (1992)). The complete CTLA-4 nucleic acidsequence can be found under GENBANK® Accession No. L15006.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including macrocyclic peptides, cytokines, and complement) that resultsin selective damage to, destruction of, or elimination from the humanbody of invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

An “adverse event” (AE) as used herein is any unfavorable and generallyunintended, even undesirable, sign (including an abnormal laboratoryfinding), symptom, or disease associated with the use of a medicaltreatment. For example, an adverse event may be associated withactivation of the immune system or expansion of immune system cells(e.g., T cells) in response to a treatment. A medical treatment may haveone or more associated AEs and each AE may have the same or differentlevel of severity. Reference to methods capable of “altering adverseevents” means a treatment regime that decreases the incidence and/orseverity of one or more AEs associated with the use of a differenttreatment regime.

As used herein, “hyperproliferative disease” refers to conditionswherein cell growth is increased over normal levels. For example,hyperproliferative diseases or disorders include malignant diseases(e.g., esophageal cancer, colon cancer, biliary cancer) andnon-malignant diseases (e.g., atherosclerosis, benign hyperplasia, andbenign prostatic hypertrophy).

As used herein, “about” or “comprising essentially of” mean within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e., the limitations of the measurement system.For example, “about” or “comprising essentially of” can mean within oneor more than one standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” can mean a rangeof up to 20%. Furthermore, particularly with respect to biologicalsystems or processes, the terms can mean up to an order of magnitude orup to 5-fold of a value. When particular values are provided in theapplication and claims, unless otherwise stated, the meaning of “about”or “comprising essentially of” should be assumed to be within anacceptable error range for that particular value.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated.

Competition Assays

The present disclosure is also directed to macrocyclic peptides that arecapable of competing with the binding of a reference anti-PD-L1 antibody(MDX-1105) by at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, and at least about 100%. Suchmacrocyclic peptides may share structural homology with one or moremacrocyclic peptides disclosed herein, including mutant, conservativesubstitution, functional substitution, and deletion forms, provided theyspecific bind to PD-L1. For example, if a macrocyclic peptide bindssubstantially to the same region of PD-L1 as a reference anti-PD-L1antibody, the macrocyclic peptide should bind to an epitope of PD-L1that at least overlaps with the PD-L1 epitope that the anti-PD-L1monoclonal antibody binds to. The overlapping region can range from oneamino acid residue to several hundred amino acid residues. Themacrocyclic peptide should then compete with and/or block the binding ofthe anti-PD-L1 monoclonal antibody to PD-L1 and thereby decrease thebinding of the anti-PD-L1 monoclonal antibody to PD-L1, preferably by atleast about 50% in a competition assay.

Anti-PD-L1 antibodies that may be used as reference antibodies forcompetition assay purposes are known in the art. For example, thefollowing representative anti-PD-L1 antibodies may be used: MDX-1105(BMS); L01X-C (Serono), L1X3 (Serono), MSB-0010718C (Serono), and PD-L1Probody (CytomX), and the PD-L1 antibodies disclosed in co-owned WO2007/005874.

Anti-PD-1 antibodies that may be used as reference antibodies forcompetition assay purposes are known in the art. For example, thefollowing representative anti-PD-1 antibodies may be used: nivolumab(BMS); 17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 each disclosed in co-owned U.S.Pat. No. 8,008,449 (BMS), MK-3475 (Merck, disclosed in U.S. Pat. No.8,168,757), and the antibodies disclosed in U.S. Pat. No. 7,488,802.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a composition, e.g.,a pharmaceutical composition, containing one or a combination ofmacrocyclic peptides of the present disclosure, formulated together witha pharmaceutically acceptable carrier. Such compositions may include oneor a combination of (e.g., two or more different) macrocyclic peptides,or immunoconjugates or bispecific molecules of the disclosure. Forexample, a pharmaceutical composition of the disclosure can comprise acombination of macrocyclic peptides (or immunoconjugates or bispecifics)that bind to different epitopes on the target antigen or that havecomplementary activities.

Pharmaceutical compositions of the disclosure also can be administeredin combination therapy, i.e., combined with other agents. For example,the combination therapy can include a macrocyclic peptide combined withat least one other anti-inflammatory or immunosuppressant agent.Examples of therapeutic agents that can be used in combination therapyare described in greater detail below in the section on uses of themacrocyclic peptides of the disclosure.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., a macrocyclicpeptide, immunoconjugate, or bispecific molecule, may be coated in amaterial to protect the compound from the action of acids and othernatural conditions that may inactivate the compound.

The pharmaceutical compounds of the disclosure may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”or “therapeutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M. et al., J.Pharm. Sci., 66:1-19 (1977)). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the disclosure also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe disclosure is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of therapeutic situation. It is especially advantageous toformulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the macrocyclic peptide, the dosage ranges fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the hostbody weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kgbody weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg. An exemplary treatment regimeentails administration once per day, twice per day, bi-weekly,tri-weekly, weekly, once every two weeks, once every three weeks, onceevery four weeks, once a month, once every 3 months or once every threeto 6 months. Preferred dosage regimens for a macrocyclic peptide of thedisclosure include 1 mg/kg body weight or 3 mg/kg body weight viaintravenous administration, with the macrocycle being given using one ofthe following dosing schedules: (i) every four weeks for six dosages,then every three months; (ii) every three weeks; (iii) 3 mg/kg bodyweight once followed by 1 mg/kg body weight every three weeks.

In some methods, two or more macrocyclic peptides with different bindingspecificities are administered simultaneously, in which case the dosageof each compound administered falls within the ranges indicated. Thecompounds are usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of macrocyclic peptide to the target antigen inthe patient. In some methods, dosage is adjusted to achieve a plasmaconcentration of about 1-1000·mu·g/ml and in some methods about25-300·mu·g/ml.

Alternatively, the macrocyclic peptide can be administered as asustained release formulation, in which case less frequentadministration is required. The dosage and frequency of administrationcan vary depending on whether the treatment is prophylactic ortherapeutic. In prophylactic applications, a relatively low dosage isadministered at relatively infrequent intervals over a long period oftime. Some patients continue to receive treatment for the rest of theirlives. In therapeutic applications, a relatively high dosage atrelatively short intervals is sometimes required until progression ofthe disease is reduced or terminated, and preferably until the patientshows partial or complete amelioration of symptoms of disease.Thereafter, the patient can be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentdisclosure employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of a macrocyclic peptide of thedisclosure preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of tumors, a “therapeuticallyeffective dosage” preferably inhibits cell growth or tumor growth by atleast about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. The ability of a compound toinhibit tumor growth and/or HIV can be evaluated in an animal modelsystem predictive of efficacy in human tumors or viral efficacy.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit, such inhibition invitro by assays known to the skilled practitioner. A therapeuticallyeffective amount of a therapeutic compound can decrease tumor size,decrease viral load, or otherwise ameliorate symptoms in a subject. Oneof ordinary skill in the art would be able to determine such amountsbased on such factors as the subject's size, the severity of thesubject's symptoms, and the particular composition or route ofadministration selected.

In another aspect, the instant disclosure provides a pharmaceutical kitof parts comprising a macrocyclic peptide and an another immumodulator,as described herein. The kit may also further comprise instructions foruse in the treatment of a hyperproliferative disease (such as cancer asdescribed herein) and/or anti-viral disease.

A composition of the present disclosure can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for macrocyclic peptides ofthe disclosure include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, a macrocyclic peptide of the disclosure can beadministered via a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Robinson, J. R.,ed., Sustained and Controlled Release Drug Delivery Systems, MarcelDekker, Inc., New York (1978).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the disclosure can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824,or 4,596,556. Examples of well-known implants and modules useful in thepresent disclosure include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medication through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the macrocyclic peptides of the disclosure canbe formulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that therapeutic compounds of the disclosure cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811,5,374,548, and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., Ranade, V. V.,J. Clin. Pharmacol., 29:685 (1989)). Exemplary targeting moietiesinclude folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low etal.); mannosides (Umezawa et al., Biochem. Biophys. Res. Commun.,153:1038 (1988)); macrocyclic peptides (Bloeman, P. G. et al., FEBSLett., 357:140 (1995); Owais, M. et al., Antimicrob. Agents Chemother.,39:180 (1995)); surfactant protein A receptor (Briscoe et al., Am. J.Physiol., 1233:134 (1995)); p 120 (Schreier et al., J. Biol. Chem.,269:9090 (1994)); see also Keinanen, K. et al., FEBS Lett., 346:123(1994); Killion, J. J. et al., Immunomethods 4:273 (1994).

Uses and Methods of the Disclosure

The macrocyclic peptides, compositions and methods of the presentdisclosure have numerous in vitro and in vivo utilities involving, forexample, detection of PD-L1 or enhancement of immune response byblockade of PD-L1. For example, these molecules can be administered tocells in culture, in vitro or ex vivo, or to human subjects, e.g., invivo, to enhance immunity in a variety of situations. Accordingly, inone aspect, the disclosure provides a method of modifying an immuneresponse in a subject comprising administering to the subject themacrocyclic peptide of the disclosure such that the immune response inthe subject is modified. Preferably, the response is enhanced,stimulated or up-regulated. In other respects, the macrocyclic peptidemay have anti-cyno, anti-mouse, and/or anti-woodchuck binding andtherapeutic activity.

As used herein, the term “subject” is intended to include human andnon-human animals. Non-human animals includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dogs, cats,cows, horses, chickens, woodchuck, amphibians, and reptiles, althoughmammals are preferred, such as non-human primates, sheep, dogs, cats,cows and horses. Preferred subjects include human patients in need ofenhancement of an immune response. The methods are particularly suitablefor treating human patients having a disorder that can be treated byaugmenting the T-cell mediated immune response. In a particularembodiment, the methods are particularly suitable for treatment ofcancer cells in vivo. To achieve antigen-specific enhancement ofimmunity, the macrocyclic peptides can be administered together with anantigen of interest. When macrocyclic peptides to PD-L1 are administeredtogether with another agent, the two can be administered in either orderor simultaneously.

The disclosure further provides methods for detecting the presence ofhuman, woodchuck, cyno, and/or mouse PD-L1 antigen in a sample, ormeasuring the amount of human, woodchuck, cyno, and/or mouse PD-L1antigen, comprising contacting the sample, and a control sample, with areference macrocyclic peptide which specifically binds to human,woodchuck, cyno, and/or mouse PD-L1, under conditions that allow forformation of a complex between the macrocycle and human, woodchuck,cyno, and/or mouse PD-L1. The formation of a complex is then detected,wherein a difference complex formation between the sample compared tothe control sample is indicative the presence of human, woodchuck, cyno,and/or mouse PD-L1 antigen in the sample.

Given the specific binding of the macrocyclic peptides of the disclosurefor PD-L1, compared to CD28, ICOS and CTLA-4, the macrocyclic peptidesof the disclosure can be used to specifically detect PD-L1 expression onthe surface of cells and, moreover, can be used to purify PD-L1 viaimmunoaffinity purification.

Cancer

Blockade of PD-1 by macrocyclic peptides can enhance the immune responseto cancerous cells in the patient. The ligand for PD-1, PD-L1, is notexpressed in normal human cells, but is abundant in a variety of humancancers (Dong et al., Nat. Med., 8:787-789 (2002)). The interactionbetween PD-1 and PD-L1 results in a decrease in tumor infiltratinglymphocytes, a decrease in T-cell receptor mediated proliferation, andimmune evasion by the cancerous cells (Dong et al., J. Mol. Med.,81:281-287 (2003); Blank et al., Cancer Immunol. Immunother., 54:307-314(2005); Konishi et al., Clin. Cancer Res., 10:5094-5100 (2004)). Immunesuppression can be reversed by inhibiting the local interaction of PD-1to PD-L1 and the effect is additive when the interaction of PD-1 toPD-L2 is blocked as well (Iwai et al., Proc. Natl. Acad. Sci.,99:12293-12297 (2002); Brown et al., J. Immunol., 170:1257-1266 (2003)).While previous studies have shown that T-cell proliferation can berestored by inhibiting the interaction of PD-1 to PD-L1, there have beenno reports of a direct effect on cancer tumor growth in vivo by blockingthe PD-1/PD-L1 interaction. In one aspect, the present disclosurerelates to treatment of a subject in vivo using a macrocyclic peptidesuch that growth of cancerous tumors is inhibited. A macrocyclic peptidemay be used alone to inhibit the growth of cancerous tumors.Alternatively, a macrocyclic peptide may be used in conjunction withother immunogenic agents, standard cancer treatments, or othermacrocyclic peptides, as described below.

Accordingly, in one embodiment, the disclosure provides a method ofinhibiting growth of tumor cells in a subject, comprising administeringto the subject a therapeutically effective amount of a macrocyclicpeptide.

Preferred cancers whose growth may be inhibited using the macrocyclicpeptides of the disclosure include cancers typically responsive toimmunotherapy. Non-limiting examples of preferred cancers for treatmentinclude melanoma (e.g., metastatic malignant melanoma), renal cellcarcinoma (e.g., clear cell carcinoma), prostate cancer (e.g., hormonerefractory prostate adenocarcinoma and castration-resistant prostatecancer), breast cancer, colorectal cancer and lung cancer (e.g.,squamous and non-squamous non-small cell lung cancer). Additionally, thedisclosure includes refractory or recurrent malignancies whose growthmay be inhibited using the macrocyclic peptides of the disclosure.

Examples of other cancers that may be treated using the methods of thedisclosure include bone cancer, pancreatic cancer, skin cancer, cancerof the head or neck, cutaneous or intraocular malignant melanoma,uterine cancer, ovarian cancer, colon cancer, rectal cancer, cancer ofthe anal region, stomach/gastric cancer, testicular cancer, uterinecancer, carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina, carcinoma of thevulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers. The present disclosure is also useful fortreatment of metastatic cancers, especially metastatic cancers thatexpress PD-L1 (Iwai et al., Int. Immunol., 17: 133-144 (2005)).

Optionally, macrocyclic peptides to PD-L1 can be combined with animmunogenic agent, such as cancerous cells, purified tumor antigens(including recombinant proteins, peptides, and carbohydrate molecules),cells, and cells transfected with genes encoding immune stimulatingcytokines (He et al., J. Immunol., 173:4919-4928 (2004)). Non-limitingexamples of tumor vaccines that can be used include peptides of melanomaantigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/ortyrosinase, or tumor cells transfected to express the cytokine GM-CSF(discussed further below).

In humans, some tumors have been shown to be immunogenic such asmelanomas. It is anticipated that by raising the threshold of T cellactivation by PD-L1 blockade, we may expect to activate tumor responsesin the host.

PD-L1 blockade is likely to be most effective when combined with avaccination protocol. Many experimental strategies for vaccinationagainst tumors have been devised (see Rosenberg, S., Development ofCancer Vaccines, ASCO Educational Book Spring: 60-62 (2000); Logothetis,C., ASCO Educational Book Spring: 300-302 (2000); Khayat, D., ASCOEducational Book Spring: 414-428 (2000); Foon, K., ASCO Educational BookSpring: 730-738 (2000); see also Restifo, N. et al., Cancer Vaccines,Chapter 61, pp. 3023-3043, in DeVita, V. et al., eds., Cancer:Principles and Practice of Oncology, Fifth Edition (1997)). In one ofthese strategies, a vaccine is prepared using autologous or allogeneictumor cells. These cellular vaccines have been shown to be mosteffective when the tumor cells are transduced to express GM-CSF. GM-CSFhas been shown to be a potent activator of antigen presentation fortumor vaccination (Dranoff et al., Proc. Natl. Acad. Sci. USA, 90:3539-3543 (1993)).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S. A., Immunity, 10:281-287 (1999)). In many cases,these tumor specific antigens are differentiated antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. PD-L1 blockade may be used in conjunctionwith a collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen may also include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim, N et al., Science,266:2011-2013 (1994)). (These somatic tissues may be protected fromimmune attack by various means). Tumor antigen may also be“neo-antigens” expressed in cancer cells because of somatic mutationsthat alter protein sequence or create fusion proteins between twounrelated sequences (i.e., bcr-abl in the Philadelphia chromosome), oridiotype from B cell tumors.

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which may be used in conjunction with PD-L1blockade is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot, R. et al.,Science, 269:1585-1588 (1995); Tamura, Y. et al., Science, 278:117-120(1997)).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle, F. et al., Nat. Med., 4:328-332 (1998)). DCs mayalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler, A. et al., Nat. Med., 6:332-336 (2000)). As amethod of vaccination, DC immunization may be effectively combined withPD-L1 blockade to activate more potent anti-tumor responses.

PD-L1 blockade may also be combined with standard cancer treatments.PD-L1 blockade may be effectively combined with chemotherapeuticregimes. In these instances, it may be possible to reduce the dose ofchemotherapeutic reagent administered (Mokyr, M. et al., Cancer Res.,58:5301-5304 (1998)). An example of such a combination is a macrocyclicpeptide in combination with decarbazine for the treatment of melanoma.Another example of such a combination is a macrocyclic peptide incombination with interleukin-2 (IL-2) for the treatment of melanoma. Thescientific rationale behind the combined use of PD-L1 blockade andchemotherapy is that cell death, that is a consequence of the cytotoxicaction of most chemotherapeutic compounds, should result in increasedlevels of tumor antigen in the antigen presentation pathway. Othercombination therapies that may result in synergy with PD-L1 blockadethrough cell death are radiation, surgery, and hormone deprivation. Eachof these protocols creates a source of tumor antigen in the host.Angiogenesis inhibitors may also be combined with PD-L1 blockade.Inhibition of angiogenesis leads to tumor cell death which may feedtumor antigen into host antigen presentation pathways.

PD-L1 blocking macrocyclic peptides can also be used in combination withbispecific macrocyclic peptides that target Fc alpha or Fc gammareceptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat.Nos. 5,922,845 and 5,837,243). Bispecific macrocyclic peptides can beused to target two separate antigens. For example anti-Fc receptor/antitumor antigen (e.g., Her-2/neu) bispecific macrocyclic peptides havebeen used to target macrophages to sites of tumor. This targeting maymore effectively activate tumor specific responses. The T cell arm ofthese responses would be augmented by the use of PD-L1 blockade.Alternatively, antigen may be delivered directly to DCs by the use ofbispecific macrocyclic peptides which bind to tumor antigen and adendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-beta (Kehrl, J. et al., J. Exp. Med.,163:1037-1050 (1986)), IL-10 (Howard, M. et al., Immunology Today,13:198-200 (1992)), and Fas ligand (Hahne, M. et al., Science,274:1363-1365 (1996)). Macrocyclic peptides to each of these entitiesmay be used in combination with anti-PD-L1 to counteract the effects ofthe immunosuppressive agent and favor tumor immune responses by thehost.

Other macrocyclic peptides which may be used to activate host immuneresponsiveness can be used in combination with anti-PD-L1. These includemolecules on the surface of dendritic cells which activate DC functionand antigen presentation. Anti-CD40 macrocyclic peptides are able tosubstitute effectively for T cell helper activity (Ridge, J. et al.,Nature, 393:474-478 (1998)) and can be used in conjunction with PD-1antibodies (Ito, N. et al., Immunobiology, 201(5):527-540 (2000)).Activating macrocyclic peptides to T cell costimulatory molecules suchas CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40 (Weinberg, A. et al.,Immunol., 164:2160-2169 (2000)), 4-1BB (Melero, I. et al., Nat. Med.,3:682-685 (1997), and ICOS (Hutloff, A. et al., Nature, 397:262-266(1999)) may also provide for increased levels of T cell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. PD-L1 blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to antigen-specific Tcells against tumor (Greenberg, R. et al., Science, 285:546-551 (1999)).These methods may also be used to activate T cell responses toinfectious agents such as CMV. Ex vivo activation in the presence ofmacrocyclic peptides may be expected to increase the frequency andactivity of the adoptively transferred T cells.

Infectious Diseases

Other methods of the disclosure are used to treat patients that havebeen exposed to particular toxins or pathogens. Accordingly, anotheraspect of the disclosure provides a method of treating an infectiousdisease in a subject comprising administering to the subject amacrocyclic peptide of the present disclosure such that the subject istreated for the infectious disease.

Similar to its application to tumors as discussed above, PD-L1 blockadecan be used alone, or as an adjuvant, in combination with vaccines, tostimulate the immune response to pathogens, toxins, and self-antigens.Examples of pathogens for which this therapeutic approach may beparticularly useful, include pathogens for which there is currently noeffective vaccine, or pathogens for which conventional vaccines are lessthan completely effective. These include, but are not limited to HIV,Hepatitis (A, B, and C), Influenza, Herpes, Giardia, Malaria (Butler, N.S. et al., Nature Immunology 13, 188-195 (2012); Hafalla, J. C. R., etal. PLOS Pathogens; Feb. 2, 2012)), Leishmania, Staphylococcus aureus,Pseudomonas Aeruginosa. PD-L1 blockade is particularly useful againstestablished infections by agents such as HIV that present alteredantigens over the course of the infections. These novel epitopes arerecognized as foreign at the time of anti-human PD-L1 administration,thus provoking a strong T cell response that is not dampened by negativesignals through PD-L1.

Some examples of pathogenic viruses causing infections treatable bymethods of the disclosure include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, cornavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods of the disclosure include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand conococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lyme disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods of the disclosure include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods of the disclosure include Entamoeba histolytica, Balantidiumcoli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondi, and Nippostrongylus brasiliensis.

In all of the above methods, PD-L1 blockade can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., interferons,agents targeting VEGF activity or VEGF-receptors, GM-CSF, G-CSF, IL-2),or bispecific antibody therapy, which provides for enhanced presentationof tumor antigens (see, e.g., Holliger, Proc. Natl. Acad. Sci. USA,90:6444-6448 (1993); Poljak, Structure, 2:1121-1123 (1994)).

Autoimmune Reactions

The macrocyclic peptides may provoke and amplify autoimmune responses.Indeed, induction of anti-tumor responses using tumor cell and peptidevaccines reveals that many anti-tumor responses involve anti-selfreactivities (depigmentation observed in anti-CTLA-4+GM-CSF-modified B16 melanoma in van Elsas et al. supra; depigmentation in Trp-2vaccinated mice (Overwijk, W. et al., Proc. Natl. Acad. Sci. USA,96:2982-2987 (1999)); autoimmune prostatitis evoked by TRAMP tumor cellvaccines (Hurwitz, A., supra (2000)), melanoma peptide antigenvaccination and vitiligo observed in human clinical trials (Rosenberg,S. A. et al., J. Immunother. Emphasis Tumor Immunol., 19(1):81-84(1996)).

Therefore, it is possible to consider using anti-PD-L1 blockade inconjunction with various self proteins in order to devise vaccinationprotocols to efficiently generate immune responses against these selfproteins for disease treatment. For example, Alzheimer's diseaseinvolves inappropriate accumulation of A.beta. peptide in amyloiddeposits in the brain; antibody responses against amyloid are able toclear these amyloid deposits (Schenk et al., Nature, 400:173-177(1999)).

Other self proteins may also be used as targets such as IgE for thetreatment of allergy and asthma, and TNF.alpha for rheumatoid arthritis.Finally, antibody responses to various hormones may be induced by theuse of the macrocycles disclosed herein. Neutralizing antibody responsesto reproductive hormones may be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors may also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-PD-L1macrocycles can be used for induction of therapeutic autoimmuneresponses to treat patients having an inappropriate accumulation ofother self-antigens, such as amyloid deposits, including A.beta. inAlzheimer's disease, cytokines such as TNF.alpha., and IgE.

Vaccines

The macrocyclic peptides may be used to stimulate antigen-specificimmune responses by coadministration of an anti-PD-1 macrocycle with anantigen of interest (e.g., a vaccine). Accordingly, in another aspectthe disclosure provides a method of enhancing an immune response to anantigen in a subject, comprising administering to the subject: (i) theantigen; and (ii) an anti-PD-1 macrocycle such that an immune responseto the antigen in the subject is enhanced. The antigen can be, forexample, a tumor antigen, a viral antigen, a bacterial antigen or anantigen from a pathogen. Non-limiting examples of such antigens includethose discussed in the sections above, such as the tumor antigens (ortumor vaccines) discussed above, or antigens from the viruses, bacteriaor other pathogens described above.

Suitable routes of administering the compositions (e.g., macrocyclicpeptides, multispecific and bispecific molecules and immunoconjugates)of the disclosure in vivo and in vitro are well known in the art and canbe selected by those of ordinary skill. For example, the compositionscan be administered by injection (e.g., intravenous or subcutaneous).Suitable dosages of the molecules used will depend on the age and weightof the subject and the concentration and/or formulation of thecomposition.

As previously described the macrocyclic peptides of the disclosure canbe co-administered with one or other more therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Thepeptide can be linked to the agent (as an immunocomplex) or can beadministered separate from the agent. In the latter case (separateadministration), the peptide can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, decarbazine and cyclophosphamide hydroxyurea which, bythemselves, are only effective at levels which are toxic or subtoxic toa patient. Cisplatin is intravenously administered as a 100 mg/dose onceevery four weeks and adriamycin is intravenously administered as a 60-75mg/ml dose once every 21 days. Co-administration of the macrocyclicpeptides of the present disclosure with chemotherapeutic agents providestwo anti-cancer agents which operate via different mechanisms whichyield a cytotoxic effect to human tumor cells. Such co-administrationcan solve problems due to development of resistance to drugs or a changein the antigenicity of the tumor cells which would render themunreactive with the peptides.

Also within the scope of the present disclosure are kits comprising thecompositions of the disclosure (e.g., macrocyclic peptides, bispecificor multispecific molecules, or immunoconjugates) and instructions foruse. The kit can further contain at least one additional reagent, or oneor more additional macrocyclic peptides of the disclosure (e.g., a humanantibody having a complementary activity which binds to an epitope inPD-L1 antigen distinct from the macrocycle). Kits typically include alabel indicating the intended use of the contents of the kit. The termlabel includes any writing, or recorded material supplied on or with thekit, or which otherwise accompanies the kit.

Combination Therapy

The combination of the macrocyclic peptides of the present disclosurewith another PD-L1 antagonist and/or other immunomodulator is useful forenhancement of an immune response against a hyperproliferative disease.For example, these molecules can be administered to cells in culture, invitro or ex vivo, or to human subjects, e.g., in vivo, to enhanceimmunity in a variety of situations. Accordingly, in one aspect, thedisclosure provides a method of modifying an immune response in asubject comprising administering to the subject a macrocyclic peptide ofthe disclosure such that the immune response in the subject is modified.Preferably, the response is enhanced, stimulated or up-regulated. Inanother embodiment, the instant disclosure provides a method of alteringadverse events associated with treatment of a hyperproliferative diseasewith an immunostimulatory therapeutic agent, comprising administering amacrocyclic peptide of the present disclosure and a subtherapeutic doseof another immunomodulator to a subject.

Blockade of PD-L1 by macrocyclic peptides can enhance the immuneresponse to cancerous cells in the patient. Cancers whose growth may beinhibited using the macrocyclic peptides of the instant disclosureinclude cancers typically responsive to immunotherapy. Representativeexamples of cancers for treatment with the combination therapy of theinstant disclosure include melanoma (e.g., metastatic malignantmelanoma), renal cancer, prostate cancer, breast cancer, colon cancerand lung cancer. Examples of other cancers that may be treated using themethods of the instant disclosure include bone cancer, pancreaticcancer, skin cancer, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, ovarian cancer, rectalcancer, cancer of the anal region, stomach cancer, testicular cancer,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers. The present disclosure is also useful fortreatment of metastatic cancers.

In certain embodiments, the combination of therapeutic agents containingat least one macrocyclic peptide discussed herein may be administeredconcurrently as a single composition in a pharmaceutically acceptablecarrier, or concurrently as separate compositions wherein each agent canbe administered sequentially. For example, a second immunomodulator anda macrocyclic peptide of the present disclosure can be administeredsequentially, such as the second immunomodulator administered first andthe macrocyclic peptide second, or the macrocyclic peptide beingadministered first and the second immunomodulator second. Furthermore,if more than one dose of the combination therapy is administeredsequentially, the order of the sequential administration can be reversedor kept in the same order at each time point of administration,sequential administrations may be combined with concurrentadministrations, or any combination thereof. For example, the firstadministration of a second immunomodulator and the macrocyclic peptidemay be concurrent, the second administration may be sequential with thesecond immunomodulator first and the macrocyclic peptide second, and thethird administration may be sequential with the macrocyclic peptidefirst and second immunomodulator second, etc. Another representativedosing scheme may involve a first administration that is sequential withthe macrocyclic peptide first and the second immunomodulator second, andsubsequent administrations may be concurrent.

Optionally, the combination of the macrocyclic peptide and a secondimmunomodulator can be further combined with an immunogenic agent, suchas cancerous cells, purified tumor antigens (including recombinantproteins, peptides, and carbohydrate molecules), cells, and cellstransfected with genes encoding immune stimulating cytokines (He et al.,J. Immunol., 173:4919-4928 (2004)). Non-limiting examples of tumorvaccines that can be used include peptides of melanoma antigens, such aspeptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, ortumor cells transfected to express the cytokine GM-CSF (discussedfurther below).

A combined PD-L1 macrocyclic peptide and a second immunomodulator can befurther combined with a vaccination protocol. Many experimentalstrategies for vaccination against tumors have been devised (seeRosenberg, S., Development of Cancer Vaccines, ASCO Educational BookSpring: 60-62 (2000); Logothetis, C., ASCO Educational Book Spring:300-302 (2000); Khayat, D., ASCO Educational Book Spring: 414-428(2000); Foon, K., ASCO Educational Book Spring: 730-738 (2000); see alsoRestifo et al., Cancer Vaccines, Chapter 61, pp. 3023-3043 in DeVita etal., eds., Cancer: Principles and Practice of Oncology, Fifth Edition(1997)). In one of these strategies, a vaccine is prepared usingautologous or allogeneic tumor cells. These cellular vaccines have beenshown to be most effective when the tumor cells are transduced toexpress GM-CSF. GM-CSF has been shown to be a potent activator ofantigen presentation for tumor vaccination (Dranoff et al., Proc. Natl.Acad. Sci. USA, 90:3539-3543 (1993)).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, Immunity, 10:281-287 (1999)). In many cases, thesetumor specific antigens are differentiation antigens expressed in thetumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. In certain embodiments, a combined PD-L1macrocyclic peptide and a second immunomodulator may be used inconjunction with a collection of recombinant proteins and/or peptidesexpressed in a tumor in order to generate an immune response to theseproteins. These proteins are normally viewed by the immune system asself-antigens and are, therefore, tolerant to them. The tumor antigenmay also include the protein telomerase, which is required for thesynthesis of telomeres of chromosomes and which is expressed in morethan 85% of human cancers and in only a limited number of somatictissues (Kim et al., Science, 266:2011-2013 (1994)). (These somatictissues may be protected from immune attack by various means). Tumorantigen may also be “neo-antigens” expressed in cancer cells because ofsomatic mutations that alter protein sequence or create fusion proteinsbetween two unrelated sequences (i.e., bcr-abl in the Philadelphiachromosome), or idiotype from B cell tumors.

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which may be used in conjunction with PD-L1macrocyclic peptide blockade is purified heat shock proteins (HSP)isolated from the tumor tissue itself. These heat shock proteins containfragments of proteins from the tumor cells and these HSPs are highlyefficient at delivery to antigen presenting cells for eliciting tumorimmunity (Suot et al., Science, 269:1585-1588 (1995); Tamura et al.,Science, 278:117-120 (1997)).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al., Nat. Med., 4:328-332 (1998)). DCs may alsobe transduced by genetic means to express these tumor antigens as well.DCs have also been fused directly to tumor cells for the purposes ofimmunization (Kugler et al., Nat. Med., 6:332-336 (2000)). As a methodof vaccination, DC immunization may be effectively further combined witha combined anti-PD-L1 macrocyclic peptide and a second immunomodulatorto activate more potent anti-tumor responses.

A combined anti-PD-L1 macrocyclic peptide and additional immunomodulatormay also be further combined with standard cancer treatments. Forexample, a combination of a macrocyclic peptide and a secondimmunomodulator may be effectively combined with chemotherapeuticregimes. In these instances, as is observed with the combination of amacrocyclic peptide and a second immunomodulator, it may be possible toreduce the dose of other chemotherapeutic reagent administered with thecombination of the instant disclosure (Mokyr et al., Cancer Res.,58:5301-5304 (1998)). An example of such a combination is a combinationof a macrocyclic peptide and a second immunomodulator further incombination with decarbazine for the treatment of melanoma. Anotherexample is a combination of a macrocyclic peptide and a secondimmunomodulatory agent further in combination with interleukin-2 (IL-2)for the treatment of melanoma. The scientific rationale behind thecombined use of PD-L1 macrocyclic peptide and another immunomodulatorwith chemotherapy is that cell death, which is a consequence of thecytotoxic action of most chemotherapeutic compounds, should result inincreased levels of tumor antigen in the antigen presentation pathway.Other combination therapies that may result in synergy with a combinedanti-PD-L1 macrocyclic peptide and additional immunomodulator throughcell death include radiation, surgery, or hormone deprivation. Each ofthese protocols creates a source of tumor antigen in the host.Angiogenesis inhibitors may also be combined with a combined PD-L1 andsecond immunomodulator. Inhibition of angiogenesis leads to tumor celldeath, which may also be a source of tumor antigen to be fed into hostantigen presentation pathways.

A combination of PD-L1 and another immunomodulator can also be used incombination with bispecific macrocyclic peptides that target Fc.alpha.or Fc.gamma. receptor-expressing effector cells to tumor cells (see,e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243). Bispecific macrocyclicpeptides can be used to target two separate antigens. For exampleanti-Fc receptor/anti tumor antigen (e.g., Her-2/neu) bispecificmacrocyclic peptides have been used to target macrophages to sites oftumor. This targeting may more effectively activate tumor specificresponses. The T cell arm of these responses would be augmented by theuse of a combined PD-L1 and a second immunomodulator. Alternatively,antigen may be delivered directly to DCs by the use of bispecificmacrocyclic peptides which bind to tumor antigen and a dendritic cellspecific cell surface marker.

In another example, a combination of a macrocyclic peptide and a secondimmunomodulator can be used in conjunction with anti-neoplasticmacrocyclic agents, such as RITUXAN® (rituximab), HERCEPTIN®(trastuzumab), BEXXAR® (tositumomab), ZEVALIN® (ibritumomab), CAMPATH®(alemtuzumab), Lymphocide (eprtuzumab), AVASTIN® (bevacizumab), andTARCEVA® (erlotinib), and the like. By way of example and not wishing tobe bound by theory, treatment with an anti-cancer antibody or ananti-cancer antibody conjugated to a toxin can lead to cancer cell death(e.g., tumor cells) which would potentiate an immune response mediatedby the second immunomodulator target or PD-L1. In an exemplaryembodiment, a treatment of a hyperproliferative disease (e.g., a cancertumor) may include an anti-cancer antibody in combination with amacrocyclic peptide and a second immunomodulator concurrently orsequentially or any combination thereof, which may potentiate ananti-tumor immune responses by the host.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofproteins, which are expressed by the tumors and which areimmunosuppressive. These include, among others, TGF-.beta. (Kehrl, J. etal., J. Exp. Med., 163:1037-1050 (1986)), IL-10 (Howard, M. et al.,Immunology Today, 13:198-200 (1992)), and Fas ligand (Hahne, M. et al.,Science, 274:1363-1365 (1996)). In another example, antibodies to eachof these entities may be further combined with a macrocyclic peptide andanother immunomodulator to counteract the effects of immunosuppressiveagents and favor anti-tumor immune responses by the host.

Other agents that may be used to activate host immune responsiveness canbe further used in combination with a macrocyclic peptide of the presentdisclosure. These include molecules on the surface of dendritic cellsthat activate DC function and antigen presentation. Anti-CD40macrocyclic peptides are able to substitute effectively for T cellhelper activity (Ridge, J. et al., Nature, 393:474-478 (1998)) and canbe used in conjunction with the macrocyclic peptides of the presentdisclosure, either alone or in combination with an anti-CTLA-4combination (Ito, N. et al., Immunobiology, 201(5):527-540 (2000)).Activating macrocyclic peptides to T cell costimulatory molecules, suchas OX-40 (Weinberg, A. et al., Immunol., 164:2160-2169 (2000)), 4-1BB(Melero, I. et al., Nat. Med., 3:682-685 (1997), and ICOS (Hutloff, A.et al., Nature, 397:262-266 (1999)) may also provide for increasedlevels of T cell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. A macrocyclic peptide of the presentdisclosure, either alone or in combination with another immunomodulator,can be used to increase the effectiveness of the donor engrafted tumorspecific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to antigen-specific Tcells against tumor (Greenberg, R. et al., Science, 285:546-551 (1999)).These methods may also be used to activate T cell responses toinfectious agents such as CMV. Ex vivo activation in the presence amacrocyclic peptide of the present disclosure, either alone or incombination with another innumomodulator, may be expected to increasethe frequency and activity of the adoptively transferred T cells.

In certain embodiments, the present disclosure provides a method foraltering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering a macrocyclic peptide of the present disclosure incombination with a subtherapeutic dose of another immunomodulator to asubject. For example, the methods of the present disclosure provide fora method of reducing the incidence of immunostimulatory therapeuticantibody-induced colitis or diarrhea by administering a non-absorbablesteroid to the patient. Because any patient who will receive animmunostimulatory therapeutic antibody is at risk for developing colitisor diarrhea induced by such treatment, this entire patient population issuitable for therapy according to the methods of the present disclosure.Although steroids have been administered to treat inflammatory boweldisease (IBD) and prevent exacerbations of IBD, they have not been usedto prevent (decrease the incidence of) IBD in patients who have not beendiagnosed with IBD. The significant side effects associated withsteroids, even non-absorbable steroids, have discouraged prophylacticuse.

In further embodiments, a macrocyclic peptide of the present disclosure,either alone or in combination with another immunomodulator, can befurther combined with the use of any non-absorbable steroid. As usedherein, a “non-absorbable steroid” is a glucocorticoid that exhibitsextensive first pass metabolism such that, following metabolism in theliver, the bioavailability of the steroid is low, i.e., less than about20%. In one embodiment of the disclosure, the non-absorbable steroid isbudesonide. Budesonide is a locally-acting glucocorticosteroid, which isextensively metabolized, primarily by the liver, following oraladministration. ENTOCORT® EC (Astra-Zeneca) is a pH- and time-dependentoral formulation of budesonide developed to optimize drug delivery tothe ileum and throughout the colon. ENTOCORT® EC is approved in the U.S.for the treatment of mild to moderate Crohn's disease involving theileum and/or ascending colon. The usual oral dosage of ENTOCORT® EC forthe treatment of Crohn's disease is 6 to 9 mg/day. ENTOCORT® EC isreleased in the intestines before being absorbed and retained in the gutmucosa. Once it passes through the gut mucosa target tissue, ENTOCORT®EC is extensively metabolized by the cytochrome P450 system in the liverto metabolites with negligible glucocorticoid activity. Therefore, thebioavailability is low (about 10%). The low bioavailability ofbudesonide results in an improved therapeutic ratio compared to otherglucocorticoids with less extensive first-pass metabolism. Budesonideresults in fewer adverse effects, including less hypothalamic-pituitarysuppression, than systemically-acting corticosteroids. However, chronicadministration of ENTOCORT® EC can result in systemic glucocorticoideffects such as hypercorticism and adrenal suppression. See Physicians'Desk Reference Supplement, 58th Edition, 608-610 (2004).

In still further embodiments, a combination PD-L1 and anotherimmunomodulator in conjunction with a non-absorbable steroid can befurther combined with a salicylate. Salicylates include 5-ASA agentssuch as, for example: sulfasalazine (AZULFIDINE®, Pharmacia & Upjohn);olsalazine (DIPENTUM®, Pharmacia & UpJohn); balsalazide (COLAZAL®, SalixPharmaceuticals, Inc.); and mesalamine (ASACOL®, Procter & GamblePharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.;ROWASA®, Solvay).

Dosage and Formulation

A suitable peptide of Formula I, or more specifically a macrocyclicpeptide described herein, can be administered to patients to treatdiabetes and other related diseases as the compound alone and or mixedwith an acceptable carrier in the form of pharmaceutical formulations.Those skilled in the art of treating diabetes can easily determine thedosage and route of administration of the compound to mammals, includinghumans, in need of such treatment. The route of administration mayinclude but is not limited to oral, intraoral, rectal, transdermal,buccal, intranasal, pulmonary, subcutaneous, intramuscular, intradermal,sublingual, intracolonic, intraocular, intravenous, or intestinaladministration. The compound is formulated according to the route ofadministration based on acceptable pharmacy practice (Fingl et al., inThe Pharmacological Basis of Therapeutics, Chapter 1, p. 1 (1975);Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co.,Easton, Pa. (1990)).

The pharmaceutically acceptable peptide compositions described hereincan be administered in multiple dosage forms such as tablets, capsules(each of which includes sustained release or timed releaseformulations), pills, powders, granules, elixirs, in situ gels,microspheres, crystalline complexes, liposomes, micro-emulsions,tinctures, suspensions, syrups, aerosol sprays and emulsions. Thecompositions described herein can also be administered in oral,intravenous (bolus or infusion), intraperitoneal, subcutaneous,transdermally or intramuscular form, all using dosage forms well knownto those of ordinary skill in the pharmaceutical arts. The compositionsmay be administered alone, but generally will be administered with apharmaceutical carrier selected on the basis of the chosen route ofadministration and standard pharmaceutical practice.

The dosage regimen for the compositions described herein will, ofcourse, vary depending upon known factors, such as the pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the species, age, sex, health, medical condition, andweight of the recipient; the nature and extent of the symptoms; the kindof concurrent treatment; the frequency of treatment; the route ofadministration, the renal and hepatic function of the patient, and theeffect desired. A physician or veterinarian can determine and prescribethe effective amount of the drug required to prevent, counter, or arrestthe progress of the disease state.

By way of general guidance, the daily oral dosage of the activeingredient, when used for the indicated effects, will range betweenabout 0.001 to 1000 mg/kg of body weight, preferably between about 0.01to 100 mg/kg of body weight per day, and most preferably between about0.6 to 20 mg/kg/day. Intravenously, the daily dosage of the activeingredient when used for the indicated effects will range between 0.001ng to 100.0 ng per min/per Kg of body weight during a constant rateinfusion. Such constant intravenous infusion can be preferablyadministered at a rate of 0.01 ng to 50 ng per min per Kg body weightand most preferably at 0.01 ng to 10.0 mg per min per Kg body weight.The compositions described herein may be administered in a single dailydose, or the total daily dosage may be administered in divided doses oftwo, three, or four times daily. The compositions described herein mayalso be administered by a depot formulation that will allow sustainedrelease of the drug over a period of days/weeks/months as desired.

The compositions described herein can be administered in intranasal formvia topical use of suitable intranasal vehicles, or via transdermalroutes, using transdermal skin patches. When administered in the form ofa transdermal delivery system, the dosage administration will, ofcourse, be continuous rather than intermittent throughout the dosageregimen.

The compositions are typically administered in a mixture with suitablepharmaceutical diluents, excipients, or carriers (collectively referredto herein as pharmaceutical carriers) suitably selected with respect tothe intended form of administration, that is, oral tablets, capsules,elixirs, aerosol sprays generated with or without propellant and syrups,and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic, pharmaceutically acceptable, inert carrier such as but notlimited to, lactose, starch, sucrose, glucose, methyl cellulose,magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, andsorbitol; for oral administration in liquid form, the oral drugcomponents can be combined with any oral, non-toxic, pharmaceuticallyacceptable inert carrier such as, but not limited to, ethanol, glycerol,and water. Moreover, when desired or necessary, suitable binders,lubricants, disintegrating agents, and coloring agents can also beincorporated into the mixture. Suitable binders include, but not limitedto, starch, gelatin, natural sugars such as, but not limited to, glucoseor beta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, and waxes. Lubricants used in these dosage formsinclude sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, and sodium chloride. Disintegrants include,but are not limited to, starch, methyl cellulose, agar, bentonite, andxanthan gum.

The compositions described herein may also be administered in the formof mixed micellar or liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles, and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine, or phosphatidylcholines. Permeationenhancers may be added to enhance drug absorption.

Since prodrugs are known to enhance numerous desirable qualities ofpharmaceuticals (i.e., solubility, bioavailability, manufacturing, etc.)the compounds described herein may be delivered in prodrug form. Thus,the subject matter described herein is intended to cover prodrugs of thepresently claimed compounds, methods of delivering the same, andcompositions containing the same.

The compositions described herein may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropyl-methacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compositionsdescribed herein may be combined with a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, andcrosslinked or amphipathic block copolymers of hydrogels.

Dosage forms (pharmaceutical compositions) suitable for administrationmay contain from about 0.01 milligram to about 500 milligrams of activeingredient per dosage unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about0.5-95% by weight based on the total weight of the composition.

Gelatin capsules may contain the active ingredient and powderedcarriers, such as lactose, starch, cellulose derivative, magnesiumstearate, and stearic acid. Similar diluents can be used to makecompressed tablets. Both tablets and capsules can be manufactured assustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose),and related sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solution for parenteral administration preferably contains awater-soluble salt of the active ingredient, suitable stabilizingagents, and if necessary, buffer substances. Antioxidizing agents suchas sodium bisulfite, sodium sulfite, or ascorbic acid, either alone orcombined, are suitable stabilizing agents. Also used are citric acid andits salts and sodium EDTA. In addition, parenteral solutions can containpreservatives, such as benzalkonium chloride, methyl- or propyl-paraben,and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington: The Scienceand Practice of Pharmacy, Nineteenth Edition, Mack Publishing Company(1995), a standard reference text in this field.

Representative useful pharmaceutical dosage forms for administration ofthe compounds described herein can be illustrated as follows:

Capsules

A large number of unit capsules can be prepared by filling standardtwo-piece hard gelatin capsules with 100 milligrams of powdered activeingredient, 150 milligrams of lactose, 50 milligrams of cellulose, and 6milligrams magnesium stearate.

Soft Gelatin Capsules

A mixture of active ingredient in a digestible oil such as soybean oil,cottonseed oil or olive oil may be prepared and injected by means of apositive displacement pump into gelatin to form soft gelatin capsulescontaining 100 milligrams of the active ingredient. The capsules shouldbe washed and dried.

Tablets

Tablets may be prepared by conventional procedures so that the dosageunit, for example is 100 milligrams of active ingredient, 0.2 milligramsof colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275milligrams of microcrystalline cellulose, 11 milligrams of starch and98.8 milligrams of lactose. Appropriate coatings may be applied toincrease palatability or delay absorption.

Injectable

An injectable formulation of a peptide composition described herein mayor may not require the use of excipients such as those that have beenapproved by regulatory bodies. These excipients include, but are notlimited to, solvents and co-solvents, solubilizing, emulsifying orthickening agents, chelating agents, antioxidants and reducing agents,antimicrobial preservatives, buffers and pH adjusting agents, bulkingagents, protectants and tonicity adjustors and special additives. Aninjectable formulation has to be sterile, pyrogen free and, in the caseof solutions, free of particulate matter.

A parenteral composition suitable for administration by injection may beprepared by stirring for example, 1.5% by weight of active ingredient ina pharmaceutically acceptable buffer that may or may not contain aco-solvent or other excipient. The solution should be made isotonic withsodium chloride and sterilized.

The abbreviations used in the present application, includingparticularly in the illustrative examples which follow, are well-knownto those skilled in the art. Some of the abbreviations used are asfollows: HOBt for hydroxybenzotriazole; HOAt for1-hydroxy-7-azabenzotriazole; DIC for N,N′-diisopropylcarbodiimide; HBTUfor 0-(benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; BOP forbenzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate; PyBOP for(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; TISor TIPS for triisopropylsilane; DMSO for dimethylsulfoxide; MeCN or ACNfor acetonitrile; DCM for dichloromethane; min for minutes; NMP forN-methylpyrrolidinone; h for hours; RT for room temperature or retentiontime (context will dictate); EtOAc for ethyl acetate; FMOC for9-fluorenylmethyloxycarbonyl; OAc for acetate; MeOH for methanol; TFAfor trifluoracetic acid; Et for ethyl; DMAP for4-(N,N-dimethylamino)pyridine; EDCI for1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EtOH for ethanol; DEA fordiethylamine; DCC for dicyclohexylcarbodiimide; DMF forN,N-dimethylformamide; EtOAc for ethyl acetate; DIEA fordiisopropylethylamine; and HATU forO-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate.

Suspension

An aqueous suspension can be prepared for oral and/or parenteraladministration so that, for example, each 5 mL contains 100 mg of finelydivided active ingredient, 20 mg of sodium carboxymethyl cellulose, 5 mgof sodium benzoate, 1.0 g of sorbitol solution, U.S.P., and 0.025 mL ofvanillin or other palatable flavoring.

Biodegradable Microparticles

A sustained-release parenteral composition suitable for administrationby injection may be prepared, for example, by dissolving a suitablebiodegradable polymer in a solvent, adding to the polymer solution theactive agent to be incorporated, and removing the solvent from thematrix thereby forming the matrix of the polymer with the active agentdistributed throughout the matrix.

Peptide Synthesis

It should be understood that the group —C(O)NH— can be oriented withinlinkers X and X′ in either of the two possible orientations (e.g., as—C(O)NH— or as —NHC(O)—) unless otherwise noted.

The description of the present disclosure herein should be construed incongruity with the laws and principals of chemical bonding. It should beunderstood that the compounds encompassed by the present disclosure arethose that are suitably stable for use as pharmaceutical agent. Forexample, in compounds of formula (I) while X is a chain of between 1 and172 atoms wherein the atoms are selected from carbon and oxygen andwherein the chain may contain one, two, three, or four groups selectedfrom —NHC(O)NH—, and —C(O)NH— embedded therein; and wherein the chain isoptionally substituted with one to six groups independently selectedfrom —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and —(CH₂)CO₂, it should beunderstood that this does not encompass compounds where multipleheteroatoms are linked to each other (i.e., —O—O— or O—NHC(O)NH—) asthese would not be considered to be stable molecules. In anotherexample, X would not encompass compounds wherein two heteroatoms areseparated only by one carbon as this would also not be considered to bestable. One of skill in the art will know what compounds would and wouldnot be stable based on the general principles of chemical bonding andstability.

Chemical synthesis of a macrocyclic peptide of the present disclosurecan be carried out using a variety of art recognized methods, includingstepwise solid phase synthesis, semi-synthesis through theconformationally-assisted re-ligation of peptide fragments, enzymaticligation of cloned or synthetic peptide segments, and chemical ligation.A preferred method to synthesize the macrocyclic peptides and analogsthereof described herein is chemical synthesis using various solid-phasetechniques such as those described in Chan, W. C. et al., eds., FmocSolid Phase Synthesis, Oxford University Press, Oxford (2000); Barany,G. et al., The Peptides: Analysis, Synthesis, Biology, Vol. 2: “SpecialMethods in Peptide Synthesis, Part A”, pp. 3-284, Gross, E. et al.,eds., Academic Press, New York (1980); and in Stewart, J. M. et al.,Solid-Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co.,Rockford, Ill. (1984). The preferred strategy is based on the Fmoc(9-Fluorenylmethyl methyl-oxycarbonyl) group for temporary protection ofthe α-amino group, in combination with the tert-butyl group fortemporary protection of the amino acid side chains (see for exampleAtherton, E. et al., “The Fluorenylmethoxycarbonyl Amino ProtectingGroup”, in The Peptides: Analysis, Synthesis, Biology, Vol. 9: “SpecialMethods in Peptide Synthesis, Part C”, pp. 1-38, Undenfriend, S. et al.,eds., Academic Press, San Diego (1987).

The peptides can be synthesized in a stepwise manner on an insolublepolymer support (also referred to as “resin”) starting from theC-terminus of the peptide. A synthesis is begun by appending theC-terminal amino acid of the peptide to the resin through formation ofan amide or ester linkage. This allows the eventual release of theresulting peptide as a C-terminal amide or carboxylic acid,respectively.

The C-terminal amino acid and all other amino acids used in thesynthesis are required to have their α-amino groups and side chainfunctionalities (if present) differentially protected such that theα-amino protecting group may be selectively removed during thesynthesis. The coupling of an amino acid is performed by activation ofits carboxyl group as an active ester and reaction thereof with theunblocked α-amino group of the N-terminal amino acid appended to theresin. The sequence of α-amino group deprotection and coupling isrepeated until the entire peptide sequence is assembled. The peptide isthen released from the resin with concomitant deprotection of the sidechain functionalities, usually in the presence of appropriate scavengersto limit side reactions. The resulting peptide is finally purified byreverse phase HPLC.

The synthesis of the peptidyl-resins required as precursors to the finalpeptides utilizes commercially available cross-linked polystyrenepolymer resins (Novabiochem, San Diego, Calif.; Applied Biosystems,Foster City, Calif.). Preferred solid supports are:4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methylbenzhydrylamine resin (Rink amide MBHA resin);9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin (Sieber amide resin);4-(9-Fmoc)aminomethyl-3,5-dimethoxyphenoxy)valeryl-aminomethyl-Merrifieldresin (PAL resin), for C-terminal carboxamides. Coupling of first andsubsequent amino acids can be accomplished using HOBt, 6-Cl-HOBt or HOAtactive esters produced from DIC/HOBt, HBTU/HOBt, BOP, PyBOP, or fromDIC/6-Cl-HOBt, HCTU, DIC/HOAt or HATU, respectively. Preferred solidsupports are: 2-Chlorotrityl chloride resin and9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin (Sieber amide resin) forprotected peptide fragments. Loading of the first amino acid onto the2-chlorotrityl chloride resin is best achieved by reacting theFmoc-protected amino acid with the resin in dichloromethane and DIEA. Ifnecessary, a small amount of DMF may be added to facilitate dissolutionof the amino acid.

The syntheses of the peptide analogs described herein can be carried outby using a single or multi-channel peptide synthesizer, such as an CEMLiberty Microwave synthesizer, or a Protein Technologies, Inc. Prelude(6 channels) or Symphony (12 channels) synthesizer.

The peptidyl-resin precursors for their respective peptides may becleaved and deprotected using any standard procedure (see, for example,King, D. S. et al., Int. J. Peptide Protein Res., 36:255-266 (1990)). Adesired method is the use of TFA in the presence of water and TIS asscavengers. Typically, the peptidyl-resin is stirred in TFA/water/TIS(94:3:3, v:v:v; 1 mL/100 mg of peptidyl resin) for 2-6 hrs at roomtemperature. The spent resin is then filtered off and the TFA solutionis concentrated or dried under reduced pressure. The resulting crudepeptide is either precipitated and washed with Et₂O or is redissolveddirectly into DMSO or 50% aqueous acetic acid for purification bypreparative HPLC.

Peptides with the desired purity can be obtained by purification usingpreparative HPLC, for example, on a Waters Model 4000 or a ShimadzuModel LC-8A liquid chromatograph. The solution of crude peptide isinjected into a YMC S5 ODS (20×100 mm) column and eluted with a lineargradient of MeCN in water, both buffered with 0.1% TFA, using a flowrate of 14-20 mL/min with effluent monitoring by UV absorbance at 220nm. The structures of the purified peptides can be confirmed byelectro-spray MS analysis.

Analytical Data:

Mass Spectrometry: “ESI-MS(+)” signifies electrospray ionization massspectrometry performed in positive ion mode; “ESI-MS(−)” signifieselectrospray ionization mass spectrometry performed in negative ionmode; “ESI-HRMS(+)” signifies high-resolution electrospray ionizationmass spectrometry performed in positive ion mode; “ESI-HRMS(−)”signifies high-resolution electrospray ionization mass spectrometryperformed in negative ion mode. The detected masses are reportedfollowing the “m/z” unit designation. Compounds with exact massesgreater than 1000 were often detected as double-charged ortriple-charged ions.

High resolution mass spectrometry (HRMS) analyses were performed on aFourier Transform Orbitrap mass spectrometer (Exactive, Thermo FisherScientific, San Jose, Calif.) using positive or negative electrosprayionization operating at 25,000 resolution (full width at half heightmaximum, FWHM). The instrument was calibrated daily according tomanufacturer's specifications resulting in mass accuracy errors <5 ppm.The operating software, Xcalibur, was used to calculate theoreticalmass-to-charge values and to process the obtained data.

Analysis LCMS Condition A:

Column: Waters BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A:water with 0.05% TFA; Mobile Phase B: Acetonitrile with 0.05% TFA;Temperature: 50° C.; Gradient: 2% B to 98% B over 1 min., then a 0.5min. hold at 98% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

Analysis LCMS Condition C:

Column: Waters BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A:water with 0.2% Formic Acid and 0.01% TFA; Mobile Phase B: Acetonitrilewith 0.2% Formic acid an 0.01% TFA; Temperature: 50° C.; Gradient: 2% Bto 80% B over 2 min., 80% B to 98% B over 0.1 minute then a 0.5 min.hold at 98% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

Analysis LCMS Condition D:

Column: Waters BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50°C.; Gradient: 0-100% B over 3 min., then a 0.75-minute hold at 100% B;Flow: 1.0 mL/min; Detection: UV at 220 nm.

Analysis LCMS Condition E:

Column: Waters BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A:5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B:95:5 acetonitrile:water with 0.1% trifluoroacetic acid; temperature: 50°C.; Gradient: 0-100% B over 3 min., then a 0.75-minute hold at 100% B;Flow: 1.11 mL/min; Detection: UV at 220 nm.

Analysis HPLC Condition B:

Column: YMC Pack ODS-AQ 3 um 150×4.6 mm; Mobile Phase A: water with 0.1%TFA; Mobile Phase B: Acetonitrile with 0.1% TFA; Temperature: 40° C.;Gradient: from 10% B to 100% B over 10 to 40 min.; Flow rate: 1 mL/min;Detection: UV at 220 nm.

General Procedures: Prelude Method A:

All manipulations were performed under automation on a Prelude peptidesynthesizer (Protein Technologies). All procedures unless noted wereperformed in a 10 or 45 mL polypropylene tube fitted with a bottom frit.The tube connects to the Prelude peptide synthesizer through both thebottom and the top of the tube. DMF and DCM can be added through the topof the tube, which washes down the sides of the tube equally. Theremaining reagents are added through the bottom of the tube and pass upthrough the frit to contact the resin. All solutions are removed throughthe bottom of the tube. “Periodic agitation” describes a brief pulse ofN₂ gas through the bottom frit; the pulse lasts approximately 5 secondsand occurs every 30 seconds. Amino acid solutions were generally notused beyond three weeks from preparation. HATU solution was used within5 days of preparation. DMF=dimethylformamide;HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium;HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate; NMM=N-methylmorpholine;Sieber=Fmoc-amino-xanthen-3-yloxy, where “3-yloxy” describes theposition and type of connectivity to the polystyrene resin. The resinused is Merrifield polymer (polystyrene) with a Sieber linker(Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading.Common amino acids used are listed below with side-chain protectinggroups indicated inside parenthesis.

Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH;Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH;Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Ile-OH;Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc[N-Me]Ala-OH;Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH;Fmoc-(D)-cis-Pro(4-OtBu)-OH; Fmoc-(D)-trans-Pro(4-OtBu)-OH; Fmoc-Sar-OH;Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH;Fmoc-Val-OH.

The procedures of “Prelude Method A” describe an experiment performed ona 0.100 mmol scale, where the scale is determined by the amount ofSieber linker bound to the resin. This scale corresponds toapproximately 140 mg of the Sieber-Merrifield resin described above. Allprocedures can be scaled beyond 0.100 mmol scale by adjusting thedescribed volumes by the multiple of the scale. Prior to amino acidcoupling, all peptide synthesis sequences began with a resin-swellingprocedure, described below as “Resin-swelling procedure”. Coupling ofamino acids to a primary amine N-terminus used the “Single-couplingprocedure” described below.

Coupling of amino acids to a secondary amine N-terminus used the“Secondary amine-coupling procedure” described below. Coupling ofchloroacetyl group to the N-terminus of the peptide is described by the“Chloroacetyl chloride coupling procedure” or “Chloroacetic acidcoupling procedure” detailed below.

Resin-Swelling Procedure:

To a 40 mL polypropylene solid-phase reaction vessel was addedMerrifield Sieber resin (140 mg, 0.100 mmol). The resin washed (swelled)three times as follows: to the reaction vessel was added DMF (5.0 mL)and DCM (5.0 mL), upon which the mixture was periodically agitated withN2 bubbling from the bottom of the reaction vessel for 10 min. beforethe solvent was drained through the frit.

Single-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 3 or 5 min. and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0mL). The mixture was periodically agitated for 3 or 5 min. and then thesolution was drained through the frit. The resin was washed successivelyfive times as follows: for each wash, DMF (4.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 60 seconds before the solution was drained through the frit. To thereaction vessel was added the amino acid (0.2M in DMF, 5.0 mL, 10 eq),then HATU or HCTU (0.2M in DMF, 5.0 mL, 10 eq), and finally NMM (0.8M inDMF, 2.5 mL, 20 eq). The mixture was periodically agitated for 60 min.,then the reaction solution was drained through the frit. The resinwashed successively four times as follows: for each wash, DMF (4.0 mL)was added through the top of the vessel and the resulting mixture wasperiodically agitated for 30 seconds before the solution was drainedthrough the frit. To the reaction vessel was added a solution of aceticanhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodicallyagitated for 10 min., then the solution was drained through the frit.The resin was washed successively four times as follows: for each wash,DMF (4.0 mL) was added through the top of the vessel and the resultingmixture was periodically agitated for 90 seconds before the solution wasdrained through the frit. The resulting resin was used directly in thenext step.

Secondary Amine-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 3 or 5 min. and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0mL). The mixture was periodically agitated for 3 or 5 min. and then thesolution was drained through the frit. The resin was washed successivelyfive times as follows: for each wash, DMF (4.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 30 seconds before the solution was drained through the frit. To thereaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq),then HATU (0.2M in DMF, 2.5 mL, 5 eq), and finally NMM (0.8M in DMF, 1.5mL, 12 eq). The mixture was periodically agitated for 300 min., then thereaction solution was drained through the frit. The resin was twicewashed as follows: for each wash, DMF (4.0 mL) was added through the topof the vessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added a solution of acetic anhydride:DIEA:DMF(10:1:89 v/v/v, 5.0 mL). The mixture was periodically agitated for 10min., then the solution was drained through the frit. The resin washedsuccessively four times as follows: for each wash, DMF (4.0 mL) wasadded through the top of the vessel and the resulting mixture wasperiodically agitated for 90 seconds before the solution was drainedthrough the frit. The resulting resin was used directly in the nextstep.

Custom Amino Acids-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 3 or 5 min. and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0mL). The mixture was periodically agitated for 3 or 5 min. and then thesolution was drained through the frit. The resin was washed successivelyfive times as follows: for each wash, DMF (4.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 30 seconds before the solution was drained through the frit. To thereaction vessel was added the amino acid (0.2M in DMF, 0.5 to 2.5 mL, 1to 5 eq), then HATU (0.2M in DMF, 0.5 to 2.5 mL, 1 to 5 eq), and finallyDIPEA (0.8M in DMF, 0.5 to 1.5 mL, 4 to 12 eq). The mixture wasperiodically agitated for 60 min. to 600 min., then the reactionsolution was drained through the frit. The resin was twice washed asfollows: for each wash, DMF (2.0 mL) was added through the top of thevessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added a solution of acetic anhydride:DIEA:DMF(10:1:89 v/v/v, 5.0 mL). The mixture was periodically agitated for 10min., then the solution was drained through the frit. The resin waswashed successively four times as follows: for each wash, DMF (4.0 mL)was added through the top of the vessel and the resulting mixture wasperiodically agitated for 90 seconds before the solution was drainedthrough the frit. The resulting resin was used directly in the nextstep.

Chloroacetyl Chloride Coupling Procedure A:

To the reaction vessel containing the resin from the previous step wasadded piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 3 min. and then the solution was drained through the frit.To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0 mL). Themixture was periodically agitated for 3 min. and then the solution wasdrained through the fit. The resin washed successively five times asfollows: for each wash, DMF (4.0 mL) was added through the top of thevessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added 3.0 mL of a solution of DIPEA (4.0 mmol, 0.699mL, 40 eq), and chloroacetyl chloride (2.0 mmol, 0.160 mL, 20 eq) inDMF. The mixture was periodically agitated for 12 to 18 hours, then thesolution was drained through the frit. The resin washed successivelythree times as follows: for each wash, DMF (4.0 mL) was added to top ofthe vessel and the resulting mixture was periodically agitated for 90seconds before the solution was drained through the frit. The resinwashed successively four times as follows: for each wash, CH₂Cl₂ (2.0mL) was added to top of the vessel and the resulting mixture wasperiodically agitated for 90 seconds before the solution was drainedthrough the frit.

Chloroacetic Acid Coupling Procedure A:

To the reaction vessel containing the resin from the previous step wasadded piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 3 min. and then the solution was drained through the frit.To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0 mL). Themixture was periodically agitated for 3 min. and then the solution wasdrained through the fit. The resin washed successively five times asfollows: for each wash, DMF (4.0 mL) was added through the top of thevessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added DMF (2.0 mL), chloroacetic acid (1.2 mmol, 113mg, 12 eq), and N,N′-Diisopropylcarbodiimide (1.2 mmol, 0.187 mL, 12eq). The mixture was periodically agitated for 12 to 18 hours, then thesolution was drained through the frit. The resin washed successivelythree times as follows: for each wash, DMF (4.0 mL) was added to top ofthe vessel and the resulting mixture was periodically agitated for 90seconds before the solution was drained through the frit. The resinwashed successively four times as follows: for each wash, CH₂Cl₂ (2.0mL) was added to top of the vessel and the resulting mixture wasperiodically agitated for 90 seconds before the solution was drainedthrough the frit.

CEM Method A:

All manipulations were performed under automation on a CEM Libertymicrowave peptide synthesizer (CEM Corporation). All procedures unlessnoted were performed in a 30 or 125 mL polypropylene tube fitted with abottom fit to a CEM Discovery microwave unit. The tube connects to theCEM Liberty synthesizer through both the bottom and the top of the tube.DMF and DCM can be added through the top and bottom of the tube, whichwashes down the sides of the tube equally. All solutions are removedthrough the bottom of the tube except while transferring resin from thetop. “Periodic bubbling” describes a brief bubbling of N₂ gas throughthe bottom frit. Amino acid solutions were generally not used beyondthree weeks from preparation. HATU solution was used within 5 days ofpreparation. DMF=dimethylformamide;HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium;HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate; DIEA/DIPEA=diisopropylethylamine;Sieber=Fmoc-amino-xanthen-3-yloxy, where “3-yloxy” describes theposition and type of connectivity to the polystyrene resin. The resinused is Merrifield polymer (polystyrene) with a Sieber linker(Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading.Common amino acids used are listed below with side-chain protectinggroups indicated inside parenthesis. Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH;Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH;Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH;Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH;Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH;Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH;Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH;Fmoc-Val-OH.

The procedures of “CEM Method A” describe an experiment performed on a0.100 mmol scale, where the scale is determined by the amount of Sieberlinker bound to the resin. This scale corresponds to approximately 140mg of the Sieber-Merrifield resin described above. All procedures can bescaled beyond 0.100 mmol scale by adjusting the described volumes by themultiple of the scale. Prior to amino acid coupling, all peptidesynthesis sequences began with a resin-swelling procedure, describedbelow as “Resin-swelling procedure”. Coupling of amino acids to aprimary amine N-terminus used the “Single-coupling procedure” describedbelow. Coupling of amino acids to a secondary amine N-terminus used the“Secondary amine-coupling procedure” described below. Coupling ofchloroacetyl group to the N-terminus of the peptide is described by the“Chloroacetyl chloride coupling procedure” or “Chloroacetic acidcoupling procedure” detailed above.

Resin-Swelling Procedure:

To 50 mL polypropylene conical tube was added Merrifield Sieber resin(140 mg, 0.100 mmol). Then DMF (7 mL) was added to the tube followed byDCM (7 mL). The resin was then transferred to the reaction vessel fromtop of the vessel. The procedure is repeated additionally two times. DMF(7 mL) was added followed by DCM (7 mL). The resin was allowed to swellwith N₂ bubbling from the bottom of the reaction vessel for 15 min.before the solvent was drained through the frit.

Standard Coupling Procedure:

To the reaction vessel containing resin from the previous step was addeda solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture wasperiodically agitated for 3 min. and then the solution was drainedthrough the frit. To the reaction vessel was added a solution ofpiperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 3 min. and then the solution was drained through the frit.The resin washed successively three times as follows: DMF (7 mL) washfrom top, followed by DMF (7 mL) wash from bottom and finally with DMF(7 mL) wash from top. To the reaction vessel was added the amino acid(0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA(2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N2 bubbling for 5min. at 75° C. for all amino acids, except Fmoc-Cys(Trt)-OH andFmoc-His(Trt)-OH which are coupled at 50° C., the reaction solution wasdrained through the frit. The resin was washed successively three timesas follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash frombottom and finally with DMF (7 mL) wash from top. To the reaction vesselwas added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0mL). The mixture was periodically bubbled for 2 min. at 65° C., then thesolution was drained through the frit. The resin washed successivelythree times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL)wash from bottom and finally with DMF (7 mL) wash from top. Theresulting resin was used directly in the next step.

Double-Couple Coupling Procedure:

To the reaction vessel containing resin from the previous step was addeda solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture wasperiodically agitated for 3 min. and then the solution was drainedthrough the frit. To the reaction vessel was added a solution ofpiperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 3 min. and then the solution was drained through the frit.The resin washed successively three times as follows: DMF (7 mL) washfrom top, followed by DMF (7 mL) wash from bottom and finally with DMF(7 mL) wash from top. To the reaction vessel was added the amino acid(0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA(2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N₂ bubbling for 5min. at 75° C. for all amino acids, except Fmoc-Cys(Trt)-OH andFmoc-His(Trt)-OH which are coupled at 50° C., the reaction solution wasdrained through the frit. The resin was washed successively three timesas follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash frombottom and finally with DMF (7 mL) wash from top. To the reaction vesselwas added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF,1.0 mL, 5 eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture wasmixed by N₂ bubbling for 5 min. at 75° C. for all amino acids, exceptFmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH which are coupled at 50° C., thereaction solution was drained through the frit. The resin washedsuccessively three times as follows: DMF (7 mL) wash from top, followedby DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash fromtop. To the reaction vessel was added a solution of aceticanhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodicallybubbled for 2 min. at 65° C., then the solution was drained through thefrit. The resin washed successively three times as follows: DMF (7 mL)wash from top, followed by DMF (7 mL) wash from bottom and finally withDMF (7 mL) wash from top. The resulting resin was used directly in thenext step.

Custom Amino Acids-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addeda solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture wasperiodically agitated for 3 min. and then the solution was drainedthrough the frit. To the reaction vessel was added a solution ofpiperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodicallyagitated for 3 min. and then the solution was drained through the frit.The resin washed successively three times as follows: DMF (7 mL) washfrom top, followed by DMF (7 mL) wash from bottom and finally with DMF(7 mL) wash from top. To the reaction vessel was added the amino acidsolution (1.25 mL to 5 mL, 2.5 eq to 10 eq) containing HATU (2.5 eq to10 eq), and finally DIPEA (2M in NMP, 0.5 mL to 1 mL, 20 eq). Themixture was mixed by N2 bubbling for 5 min. to 2 hours at 25° C. to 75°C., then the reaction solution was drained through the frit. The resinwashed successively three times as follows: DMF (7 mL) wash from top,followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) washfrom top. To the reaction vessel was added a solution of aceticanhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodicallybubbled for 2 min. at 65° C., then the solution was drained through thefrit. The resin washed successively three times as follows: DMF (7 mL)wash from top, followed by DMF (7 mL) wash from bottom and finally withDMF (7 mL) wash from top. The resulting resin was used directly in thenext step.

N-methylation on-resin (Turner, R. A.; Hauksson, N. E.; Gipe, J. H.;Lokey, R. S. Org. Lett. 2013, 15(19), 5012-5015):

All manipulations were performed manually unless noted. The procedure of“N-methylation on-resin” describes an experiment performed on a 0.100mmol scale, where the scale is determined by the amount of Sieber linkerbound to the resin that was used to generate the peptide. This scale isnot based on a direct determination of the quantity of peptide used inthe procedure. The procedure can be scaled beyond 0.100 mmol scale byadjusting the described volumes by the multiple of the scale.

The resin was transferred into a 25 mL syringe equipped with a frit. Tothe resin was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture wasshaken for 3 min. and then the solution was drained through the frit.The resin washed 3 times with DMF (4.0 mL). To the reaction vessel wasadded piperidine:DMF (20:80 v/v, 4.0 mL). The mixture was shaken for 3min. and then the solution was drained through the frit. The resinwashed successively six times as follows: 3 times DMF (4.0 mL) was addedand the resulting mixture was shaken for 3 seconds before the solutionwas drained through the fit followed by 3 addition of DCM (4.0 mL) andthe resulting mixture was shaken for 3 seconds before the solution wasdrained through the frit.

The resin was suspended in DMF (2.0 mL) and ethyl trifluoroacetate(0.119 ml, 1.00 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (0.181 ml,1.20 mmol). The mixture was put on a shaker for 60 min. The solution wasdrained through the frit. The resin washed successively six times asfollows: 3 times DMF (4.0 mL) was added and the resulting mixture wasshaken for 3 seconds before the solution was drained through the fritfollowed by 3 addition of DCM (4.0 mL) and the resulting mixture wasshaken for 3 seconds before the solution was drained through the frit.The resin washed 3 times with dry THF (2.0 mL) to remove any residualwater. In an oven dried 4.0 mL vial is added THF (1.0 mL),triphenylphosphine (131 mg, 0.500 mmol) on dry 4 Å molecular sieves (20mg). The turbid solution is transferred on the resin and isopropylazodicarboxylate (0.097 mL, 0.5 mmol) is added slowly. The resin isshaken for 15 min. The solution was drained through the fit and theresin washed with 3 times with dry THF (2.0 mL) to remove any residualwater. In an oven dried 4.0 mL vial is added THF (1.0 mL),triphenylphosphine (131 mg, 0.500 mmol) on dry 4 Å molecular sieves (20mg). The turbid solution is transferred on the resin and diisopropylazodicarboxylate (0.097 mL, 0.5 mmol) is added slowly. The resin isshaken for 15 min. The solution was drained through the frit. The resinwashed successively six times as follows: 3 times DMF (4.0 mL) was addedand the resulting mixture was shaken for 3 seconds before the solutionwas drained through the fit followed by 3 addition of DCM (4.0 mL) andthe resulting mixture was shaken for 3 seconds before the solution wasdrained through the frit.

The resin was suspended in Ethanol (1.0 mL) and THF (1.0 mL) and sodiumborohydride (37.8 mg, 1.000 mmol) was added. The mixture was mixed on ashaker for 30 min. Solution was drained through the frit and the resinwashed successively six times as follows: 3 times DMF (4.0 mL) was addedand the resulting mixture was shaken for 3 seconds before the solutionwas drained through the frit followed by 3 addition of DCM (4.0 mL) andthe resulting mixture was shaken for 3 seconds before the solution wasdrained through the frit.

Global Deprotection Method B:

All manipulations were performed manually unless noted. The procedure of“Global Deprotection Method B” describes an experiment performed on a0.100 mmol scale, where the scale is determined by the amount of Sieberlinker bound to the resin. The procedure can be scaled beyond 0.100 mmolscale by adjusting the described volumes by the multiple of the scale. A“deprotection solution” was prepared using trifluoroaceticacid:triisopropylsilane:dithiothreitol (94:3:3 v:v:w). The resin wasremoved from the reaction vessel and transferred to a 25 mL syringeequipped with a frit. To the syringe was added the “deprotectionsolution” (5.0 mL). The mixture was mixed in a shaker for 5 min. Thesolution was filtered through and diluted in diethyl ether (30 mL). Theprecipitated solid was centrifuged for 3 min. The supernatant solutionwas decanted and the solid was re-suspended in diethyl ether (25 mL).The suspension was centrifuged for 3 min. The supernatant was decantedand the remaining solid was suspended diethyl ether (25 mL). Thesuspension was centrifuged for 3 min. The supernatant was decanted andthe remaining solid was dried under high vacuum. The crude peptide wasobtained as a white to off-white solid.

Cyclization Method C:

All manipulations were performed manually unless noted. The procedure of“Cyclization Method C” describes an experiment performed on a 0.100 mmolscale, where the scale is determined by the amount of Sieber linkerbound to the resin that was used to generate the peptide. This scale isnot based on a direct determination of the quantity of peptide used inthe procedure. The procedure can be scaled beyond 0.100 mmol scale byadjusting the described volumes by the multiple of the scale. The crudepeptide solids were dissolved in a solution of acetonitrile:aqueous 0.1Mammonium bicarbonate buffer (11 mL:24 mL), and the solution was thencarefully adjusted to pH=8.5-9.0 using aqueous NaOH (1.0 M). Thesolution was then mixed using a shaker for 12 to 18 hours. The reactionsolution was concentrated and the residue was then dissolved inacetonitrile:water. This solution was subjected to reverse-phase HPLCpurification to afford the desired cyclic peptide.

Preparation of Example 3214

Example 3214 was prepared following the general synthetic sequencedescribed below.

To a 40 mL polypropylene solid-phase reaction vessel was added Sieberresin (140 mg, 0.100 mmol), and the reaction vessel was placed on thePrelude peptide synthesizer. The following procedures were thenperformed sequentially:

“Prelude Method A: Resin-swelling procedure” was followed;“Prelude Method A: Single-coupling procedure” was followed withFmoc-Lys(Boc)-OH;“Prelude Method A: Single-coupling procedure” was followed withFmoc-Gly-OH;“Prelude Method A: Single-coupling procedure” was followed withFmoc-Gly-OH;“Prelude Method A: Single-coupling procedure” was followed withFmoc-Gly-OH;“Prelude Method A: Single-coupling procedure” was followed withFmoc-Cys(Trt)-OH;“Prelude Method A: Single-coupling procedure” was followed withFmoc-Leu-OH;“Prelude Method A: Single-coupling procedure” was followed withFmoc-Tyr(tBu)-OH;“Prelude Method A: Single-coupling procedure” was followed withFmoc-Trp(Boc)-OH;“Prelude Method A: Single-coupling procedure” was followed withFmoc-[N-Me]Glu(OtBu)-OH;“Prelude Method A: Secondary amine-coupling procedure” was followed withFmoc-Tyr(tBu)-OH for 6 h;“Prelude Method A: Single-coupling procedure” was followed withFmoc-[N-Me]Phe-OH;“Prelude Method A: Secondary amine-coupling procedure” was followed withFmoc-Val-OH for 6 h;“Prelude Method A: Single-coupling procedure” was followed withFmoc-Asp(OtBu)-OH;“Prelude Method A: Single-coupling procedure” was followed withFmoc-cis-(D)-Pro(4-OH)—OH;“Prelude Method A: Secondary amine-coupling procedure” was followed withFmoc-[N-Me]Nle-OH for 6 h;“Prelude Method A: Secondary amine-coupling procedure” was followed withFmoc-[N-Me]Phe-OH for 6 h;“Prelude Method A: Secondary amine-coupling procedure” was followed withFmoc-Phe(3,4,5-tri-F)—OH for 6 h;“Prelude Method A: Chloroacetyl chloride coupling procedure A” wasfollowed;“Global Deprotection Method B” was followed;“Cyclization Method C” was followed.

The crude material was purified via preparative LC/MS with the followingConditions: Column: Phenomenex Luna 20×250 5u particles; Mobile Phase A:water with 0.1% TFA; Mobile Phase B: Acetonitrile with 0.1% TFA;Gradient: 35-95% B over 50 min., then a 5-minute hold at 95% B; Flow: 15mL/min. Fractions containing the desired product were combined and driedvia centrifugal evaporation. The yield of the product was 5.3 mg, andits estimated purity by “Analysis HPLC Condition B” was 80% using agradient of 35% to 80% buffer B in A over 30 min. Analysis LCMSCondition A: Retention time=1.33 min; ESI-MS(+) m/z 1104.1 (M+2H).ESI-HRMS(+) m/z: Calculated: 1103.5019 (M+2H); Found: 1103.5034 (M+2H).

Preparation of Example 3215

The compound from Example 3214 (4.7 mg, 2.130 μmol) was dissolved in 0.4mL DMF/ACN (1:1). DIEA (3.72 μl, 0.021 mmol) was added, followed by a0.9 mL solution of Alexa-5-SDP ester (2.93 mg, 3.5 μmol, MolecularProbes, A30052) in DMF/CH₃CN/DMSO (1:1:1). The reaction was stirred atroom temperature for 16 h. The crude material was purified viapreparative LC/MS using the following conditions: Column: YMC ODS-AQ100×10 mm S-5 um 12 nm; Mobile Phase A: water with 0.1% TFA; MobilePhase B: Acetonitrile with 0.1% TFA; Gradient: 25-75% B over 50 min.,then a 5-minute hold at 75% B; Flow: 15 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of product was 0.75 mg, and its estimated purity by “Analysis HPLCCondition B” was 96% using a gradient of 35% to 65% buffer B over 30min. Analysis LCMS Condition A: Retention time=1.39 min; ESI-MS(+) m/z1362.5 (M+2H).

Preparation of Example 3619

Example 3619 was prepared following the general synthetic sequencedescribed below.

To a 50 mL polypropylene tube was added Sieber resin (350 mg, 0.250mmol), and the tube was placed on the CEM Liberty microwave peptidesynthesizer. The following procedures were then performed sequentially:

“CEM Method A: Resin-swelling procedure” was followed;“CEM Method A: Standard coupling procedure” was followed withFmoc-Lys-OH;“CEM Method A: Standard coupling procedure” was followed withFmoc-Gly-OH;“CEM Method A: Standard coupling procedure” was followed withFmoc-Gly-OH;“CEM Method A: Standard coupling procedure” was followed withFmoc-Gly-OH;“CEM Method A: Standard coupling procedure” was followed withFmoc-Cys(Trt)-OH;“CEM Method A: Standard coupling procedure” was followed withFmoc-Leu-OH;“CEM Method A: Standard coupling procedure” was followed withFmoc-Tyr(tBu)-OH;“CEM Method A: Standard coupling procedure” was followed withFmoc-Trp(tBu)-OH;“CEM Method A: Standard coupling procedure” was followed withFmoc-[N-Me]Glu-OH;“CEM Method A: Secondary amine-coupling procedure” was followed withFmoc-Tyr(tBu)-OH;“CEM Method A: Standard coupling procedure” was followed withFmoc-[N-Me]Phe-OH;“CEM Method A: Custom amino acids-coupling procedure” was followed withFmoc-Val-OH using 10 eq for 10 min at 75° C., followed by 2 hours atroom temperature;“CEM Method A: Standard coupling procedure” was followed withFmoc-Asp(OtBu)-OH;“CEM Method A: Standard coupling procedure” was followed withFmoc-Sar-OH;“CEM Method A: Custom amino acids-coupling procedure” was followed withFmoc-[N-Me]Nle-OH using 5 eq for 10 mins;“CEM Method A: Custom amino acids-coupling procedure” was followed withFmoc-[N-Me]Phe-OH using 5 eq for 10 mins;“CEM Method A: Custom amino acids-coupling procedure” was followed withFmoc-Phe-OH using 5 eq for 10 mins;“Prelude Method A: Chloroacetyl chloride coupling procedure A” wasfollowed,“Global Deprotection Method B” was followed and “Cyclization Method C”was followed.The crude material was purified via preparative HPLC with the followingconditions: Column: Phenomenex Luna 5u C18(2) 250×21.2 AXIA, 100A Ser.#520221-1; Mobile Phase A: 0.1% TFA in water; Mobile Phase B: 0.1% TFAin acetonitrile; Gradient: 35-75% B over 40 min., then a 5-minutegradient up to 85% B; Flow: 15 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation andlyophilization. The yield of product was 12.9 mg, and its estimatedpurity by LCMS analysis was 98% using “Analysis LCMS conditions A andC”. Analysis LCMS Condition A: Retention time=1.29 min; ESI-MS(+) m/z1056.1 (M+2H). Analysis LCMS Condition C: Retention time=1.33 min;ESI-MS(+) m/z 1055.7 (M+2H); ESI-HRMS(+) m/z: Calculated: 1056.0077(M+2H); Found: 1056.0077 (M+2H).

Preparation of Example 3620

The peptide product from Example 3619 (8.0 mg, 3.79 μmol) was dissolvedinto 40 μL of DMF and 20 μL of acetonitrile. To this solution was added2,5-dioxopyrrolidin-1-yl17-oxo-21-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10,13-tetraoxa-16-azahenicosan-1-oate(2.231 mg, 3.79 μmol) and N,N-Diisopropylethylamine (6.60 μl, 0.038mmol). The solution was stirred for 6 h. The crude material was purifiedvia preparative HPLC with the following conditions: Column: PhenomenexLuna 5u C18(2) 250×21.2 AXIA, 100A Ser. #520221-1; Mobile Phase A: 0.1%TFA in water; Mobile Phase B: 0.1% TFA in acetonitrile; Gradient: 35-75%B over 40 min., then a 5-minute gradient up to 85% B; Flow: 15 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation and further dried by lyophilization.

The yield of product was 4.4 mg, and its estimated purity by LCMSanalysis was 99.5% using “Analysis LCMS conditions A and C”. AnalysisLCMS Condition A: Retention time=1.34 min; ESI-MS(+) m/z 1292.8 (M+2H).Analysis LCMS Condition C: Retention time=1.56 min; ESI-MS(+) m/z 1292.4(M+2H); ESI-HRMS(+) m/z: Calculated: 1292.1206 (M+2H); Found: 1292.1219(M+2H).

Preparation of Example 3621

Example 3621 was prepared following the general synthetic sequencedescribed for the preparation of Example 3619, composed of the followingprocedures: “CEM Method A: Resin-swelling procedure”, “CEM Method A:Standard coupling procedure”, “CEM Method A: Secondary amine-couplingprocedure”, “CEM Method A: Custom amino acids-coupling procedure”,“Chloroacetic acid coupling procedure A”, “Global Deprotection MethodB”, and “Cyclization Method C”. The crude material was purified viapreparative HPLC with the following conditions: Column: Phenomenex Luna5u C18(2) 250×21.2 AXIA, 100A Ser. #520221-1; Mobile Phase A: 0.1% TFAin water; Mobile Phase B: 0.1% TFA in acetonitrile; Gradient: 35-75% Bover 40 min., then a 5-minute gradient up to 85% B; Flow: 15 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation, followed by lyophilization. The peptide (6.0mg, 2.84 μmol) was dissolved in 100 μL of DMF. To this solution wasadded 2,5-dioxopyrrolidin-1-yl2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oate (1.950mg, 2.84 μmol) and N,N-Diisopropylethylamine (4.95 μl, 0.028 mmol). Thesolution was stirred for 3 h. The crude material was purified viapreparative HPLC with the following conditions: Column: Phenomenex Luna5u C18(2) 250×21.2 AXIA, 100A Ser. #520221-1; Mobile Phase A: 0.1% TFAin water; Mobile Phase B: 0.1% TFA in acetonitrile; Gradient: 35-75% Bover 40 min., then a 5-minute gradient up to 85% B; Flow: 15 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation, followed by lyophilization. The yield of theproduct was 4.0 mg, and its estimated purity by LCMS analysis was 98%using “Analysis LCMS conditions A and C”. Analysis LCMS Condition A:Retention time=1.34 min; ESI-MS(+) m/z 1341.0 (M+2H). Analysis LCMSCondition C: Retention time=1.62 min; ESI-MS(+) m/z 1341.4 (M+2H).

Preparation of Example 3622

Example 3622 was prepared following the general synthetic sequencedescribed for the preparation of Example 3619, composed of the followingprocedures: “CEM Method A: Resin-swelling procedure”, “CEM Method A:Standard coupling procedure”, “CEM Method A: Secondary amine-couplingprocedure”, “CEM Method A: Custom amino acids-coupling procedure”,“Chloroacetic acid coupling procedure A”, “Global Deprotection MethodB”, and “Cyclization Method C”. The crude material was purified viapreparative HPLC with the following conditions: Column: Phenomenex Luna5u C18(2) 250×21.2 AXIA, 100A Ser. #520221-1; Mobile Phase A: 0.1% TFAin water; Mobile Phase B: 0.1% TFA in acetonitrile; Gradient: 35-75% Bover 40 min., then a 5-minute gradient up to 85% B; Flow: 15 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation, followed by lyophilization. The peptide (6.0mg, 2.84 μmol) was dissolved in 100 μL of DMF. To this solution wasadded2,5-ioxopyrrolidin-1-yl-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,134,137,140,143,146-nonatetracontaoxanonatetracontahectan-149-oate(6.58 mg, 2.84 μmol) and N,N-Diisopropylethylamine (4.95 μl, 0.028mmol). The solution was stirred for 3 h. The crude material was purifiedvia preparative HPLC with the following conditions: Column: PhenomenexLuna 5u C18(2) 250×21.2 AXIA, 100A Ser. #520221-1; Mobile Phase A: 0.1%TFA in water; Mobile Phase B: 0.1% TFA in acetonitrile; Gradient: 35-75%B over 40 min., then a 5-minute gradient up to 85% B; Flow: 15 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation, followed by lyophilization. The yield of theproduct was 3.5 mg, and its estimated purity by LCMS analysis was 96%using “Analysis LCMS conditions A and C”. Analysis LCMS Condition A:Retention time=1.39 min; ESI-MS(+) m/z 1078.7 (M+4H). Analysis LCMSCondition C: Retention time=1.63 min; ESI-MS(+) m/z 1078.5 (M+4H).

Preparation of Example 3623

Example 3623 was prepared following the general synthetic sequencedescribed for the preparation of Example 3619, composed of the followingprocedures: “CEM Method A: Resin-swelling procedure”, “CEM Method A:Standard coupling procedure”, “CEM Method A: Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method B”, and “Cyclization Method C”. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B:95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Gradient: 10-60%B over 25 min., then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation, followed by lyophilization.

The yield of the product was 28 mg, and its estimated purity by LCMSanalysis was 98% using “Analysis LCMS conditions D and E”. Analysis LCMSCondition D: Retention time=1.69 min; ESI-MS(+) m/z 1088.1 (M+2H).Analysis LCMS Condition E: Retention time=1.80 min; ESI-MS(+) m/z 1088.2(M+2H). ESI-HRMS(+) m/z: Calculated: 1087.5070 (M+2H); Found: 1087.5062(M+2H).

Preparation of Example 3624

The peptide product from Example 3623 (8.0 mg, 3.68 μmol) was dissolvedin 100 μL of DMF. To this solution was added 2,5-dioxopyrrolidin-1-yl2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oate (2.78mg, 4.05 μmol) and N,N-Diisopropylethylamine (6.41 μl, 0.037 mmol). Thesolution was stirred for 3 h. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:waterwith 0.1% trifluoroacetic acid; Gradient: 10-60% B over 25 min., then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of product was 2.5 mg, and its estimated purity by LCMS analysiswas 94% using “Analysis LCMS conditions D and E”. Analysis LCMSCondition D: Retention time=1.71 min; ESI-MS(+) m/z 1390.2 (M+2H+2H₂O);Analysis LCMS Condition E: Retention time=1.89 min; ESI-MS(+) m/z 1372.9(M+2H); ESI-HRMS(+) m/z: Calculated: 1372.6696 (M+2H); Found: 1372.6729(M+2H).

Preparation of Example 3625

The peptide product from Example 3623 (8.0 mg, 3.68 μmol) was dissolvedin 100 μL of DMF. To this solution was added149-((2,5-dioxopyrrolidin-1-yl)oxy)-149-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,134,137,140,143,146-nonatetracontaoxanonatetracontahectan-56-ium(9.38 mg, 4.05 μmol) and N,N-Diisopropylethylamine (6.41 μl, 0.037mmol). The solution was stirred for 3 h. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: WatersXBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 10-60% Bover 25 min., then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of product was 3.4 mg, and its estimated purityby LCMS analysis was 91% using “Analysis LCMS conditions D and E”.Analysis LCMS Condition D: Retention time=1.76 min; ESI-MS(+) m/z 1112.0(M+4H+4H₂O). Analysis LCMS Condition E: Retention time=1.91 min;ESI-MS(+) m/z 1094.8 (M+4H).

Preparation of Example 3626

The peptide product from Example 3623 (8.0 mg, 3.68 μmol) was dissolvedin 100 μL of DMF. To this solution was added 2,5-dioxopyrrolidin-1-yl17-oxo-21-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10,13-tetraoxa-16-azahenicosan-1-oate(2.382 mg, 4.05 μmol) and N,N-Diisopropylethylamine (6.41 μl, 0.037mmol). The solution was stirred for 3 h. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: WatersXBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile:water with 0.1% trifluoroacetic acid; Gradient: 10-60% Bover 25 min., then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 2.7 mg, and its estimatedpurity by LCMS analysis was 92% using “Analysis LCMS conditions D andE”. Analysis LCMS Condition D: Retention time=1.65 min; ESI-MS(+) m/z1323.8 (M+2H). Analysis LCMS Condition E: Retention time=1.81 min;ESI-MS(+) m/z 1324.6 (M+2H). ESI-HRMS(+) m/z: Calculated: 1324.1168(M+2H) Found: 1324.1180 (M+2H).

Analytical Data:

Mass Spectrometry: “ESI-MS(+)” signifies electrospray ionization massspectrometry performed in positive ion mode; “ESI-MS(−)” signifieselectrospray ionization mass spectrometry performed in negative ionmode; “ESI-HRMS(+)” signifies high-resolution electrospray ionizationmass spectrometry performed in positive ion mode; “ESI-HRMS(−)”signifies high-resolution electrospray ionization mass spectrometryperformed in negative ion mode. The detected masses are reportedfollowing the “m/z” unit designation. Compounds with exact massesgreater than 1000 were often detected as double-charged ortriple-charged ions.

Analysis Condition A:

Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50°C.; Gradient: 0% B, 0-100% B over 3 minutes, then a 0.5-minute hold at100% B; Flow: 1 mL/min; Detection: UV at 220 nm.

Analysis Condition B:

Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 methanol:water with 10 mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10 mM ammonium acetate; Temperature: 50° C.;Gradient: 0% B, 0-100% B over 3 minutes, then a 0.5-minute hold at 100%B; Flow: 0.5 mL/min; Detection: UV at 220 nm.

Analysis Condition C:

Column: Waters Aquity BEH C18 2.1×50 mm 1.7 μm particles; Mobile PhaseA: water with 0.05% TFA; Mobile Phase B: acetonitrile with 0.05% TFA;Temperature: 40° C.; Gradient: 0% B, 0-100% B over 3 minutes, then a0.5-minute hold at 100% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

Analysis Condition D:

Column: Waters Aquity BEH C18 2.1×50 mm 1.7 μm particles; Mobile PhaseA: water with 0.05% TFA; Mobile Phase B: methanol with 0.05% TFA;Temperature: 40° C.; Gradient: 0% B, 0-100% B over 3 minutes, then a0.5-minute hold at 100% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

General Procedures: Prelude Method A:

All manipulations were performed under automation on a Prelude peptidesynthesizer (Protein Technologies). All procedures unless noted wereperformed in a 10 mL polypropylene tube fitted with a bottom frit; wherethe scale of the reaction exceeded 0.100 mmol, a 40 mL polypropylenetube fitted with a bottom frit was used. The tube connects to a thePrelude peptide synthesizer through both the bottom and the top of thetube. DMF and DCM can be added through the top of the tube, which washesdown the sides of the tube equally. The remaining reagents are addedthrough the bottom of the tube and pass up through the fit to contactthe resin. All solutions are removed through the bottom of the tube.“Periodic agitation” describes a brief pulse of N2 gas through thebottom frit; the pulse lasts approximately 5 seconds and occurs every 30seconds. Chloroacetyl chloride solutions in DMF were used within 24 h ofpreparation. Amino acid solutions were generally not used beyond threeweeks from preparation. HATU solutions were used within 5 days ofpreparation. DMF=dimethylformamide;HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate; DIPEA=diisopropylethylamine;Rink=(2,4-dimethoxyphenyl)(4-alkoxyphenyl)methanamine, where “4-alkoxy”describes the position and type of connectivity to the polystyreneresin. Unless noted otherwise, the resin used is Merrifield polymer(polystyrene) with a Rink linker (Fmoc-protected at nitrogen); 100-200mesh, 1% DVB, 0.56 mmol/g loading. Common amino acids used are listedbelow with side-chain protecting groups indicated inside parenthesis.Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH;Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH;Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH;Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH;Fmoc-[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH;Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH;Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

The procedures of “Prelude Method A” describe an experiment performed ona 0.100 mmol scale, where the scale is determined by the amount of Rinklinker bound to the resin. This scale corresponds to approximately 178mg of the Rink-Merrifield resin described above. All procedures can bescaled beyond 0.100 mmol scale by adjusting the described volumes by themultiple of the scale. Prior to amino acid coupling, all peptidesynthesis sequences began with a resin-swelling procedure, describedbelow as “Resin-swelling procedure”. Coupling of amino acids to aprimary amine N-terminus used the “Single-coupling procedure” describedbelow. Coupling of amino acids to a secondary amine N-terminus used the“Double-coupling procedure” described below. Coupling ofchloroacetylchloride to the N-terminus of the peptide is described bythe “Chloroacetyl chloride coupling procedure” detailed below.

Resin-Swelling Procedure:

The resin washed (swelled) three times as follows: to the reactionvessel was added DMF (2.0 mL), upon which the mixture was periodicallyagitated for 10 minutes before the solvent was drained through the frit.

Single-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0mL). The mixture was periodically agitated for 3 minutes and then thesolution was drained through the frit. The resin washed successively sixtimes as follows: for each wash, DMF (2.0 mL) was added through the topof the vessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added the amino acid (0.2M in DMF, 1.0 mL, 2 eq),then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA (0.8M in DMF,0.5 mL, 4 eq). The mixture was periodically agitated for 15 minutes,then the reaction solution was drained through the frit. The resinwashed successively four times as follows: for each wash, DMF (2.0 mL)was added through the top of the vessel and the resulting mixture wasperiodically agitated for 30 seconds before the solution was drainedthrough the frit. To the reaction vessel was added acetic anhydride (2.0mL). The mixture was periodically agitated for 10 minutes, then thesolution was drained through the frit. The resin washed successivelyfour times as follows: for each wash, DMF (2.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 90 seconds before the solution was drained through the frit. Theresulting resin was used directly in the next step.

Double-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0mL). The mixture was periodically agitated for 3 minutes and then thesolution was drained through the frit. The resin washed successively sixtimes as follows: for each wash, DMF (2.0 mL) was added through the topof the vessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added the amino acid (0.2M in DMF, 1.0 mL, 2 eq),then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA (0.8M in DMF,0.5 mL, 4 eq). The mixture was periodically agitated for 15 minutes,then the reaction solution was drained through the frit. The resin wastwice washed as follows: for each wash, DMF (2.0 mL) was added throughthe top of the vessel and the resulting mixture was periodicallyagitated for 30 seconds before the solution was drained through thefrit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.0mL, 2 eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA(0.8M in DMF, 0.5 mL, 4 eq). The mixture was periodically agitated for15 minutes, then the reaction solution was drained through the frit. Theresin was twice washed as follows: for each wash, DMF (2.0 mL) was addedthrough the top of the vessel and the resulting mixture was periodicallyagitated for 30 seconds before the solution was drained through thefrit. To the reaction vessel was added acetic anhydride (2.0 mL). Themixture was periodically agitated for 10 minutes, then the solution wasdrained through the frit. The resin washed successively four times asfollows: for each wash, DMF (2.0 mL) was added through the top of thevessel and the resulting mixture was periodically agitated for 90seconds before the solution was drained through the frit. The resultingresin was used directly in the next step.

Chloroacetyl Chloride Coupling Procedure:

To the reaction vessel containing the resin from the previous step wasadded piperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0mL). The mixture was periodically agitated for 3 minutes and then thesolution was drained through the frit. The resin washed successively sixtimes as follows: for each wash, DMF (2.0 mL) was added through the topof the vessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added DIPEA (0.8M in DMF, 3.0 mL, 24 eq), thenchloroacetyl chloride (0.8M in DMF, 1.65 mL, 13.2 eq). The mixture wasperiodically agitated for 30 minutes, then the solution was drainedthrough the frit. The resin washed successively three times as follows:for each wash, DMF (2.0 mL) was added to top of the vessel and theresulting mixture was periodically agitated for 90 seconds before thesolution was drained through the frit. The resin washed successivelyfour times as follows: for each wash, CH₂Cl₂ (2.0 mL) was added to topof the vessel and the resulting mixture was periodically agitated for 90seconds before the solution was drained through the frit. The resultingresin was placed under a N₂ stream for 15 minutes.

Symphony Method A:

This collection of procedures is identical that of “Prelude Method A”except as noted. For all procedures a Symphony X peptide synthesizer(Protein Technologies) was used instead of a Prelude peptide synthesizerand all reagents were added through the top of the reaction vessel.

Resin-Swelling Procedure:

This procedure is identical to “Prelude Method A: Resin-swellingprocedure”.

Single-Coupling Procedure:

This procedure is identical to “Prelude Method A: Single-couplingprocedure” except that the concentration of DIPEA solution was 0.4M and1.0 mL of this solution was delivered to the reaction.

Double-Coupling Procedure:

This procedure is identical to “Prelude Method A: Double-couplingprocedure” except that the concentration of DIPEA solution was 0.4M and1.0 mL of this solution was delivered to the reaction.

Chloroacetyl Chloride Coupling Procedure:

This procedure is identical to “Prelude Method A: Chloroacetyl chloridecoupling procedure”.

Global Deprotection Method A:

All manipulations were performed manually unless noted. The procedure of“Global Deprotection Method A” describes an experiment performed on a0.100 mmol scale, where the scale is determined by the amount of Rinklinker bound to the resin. The procedure can be scaled beyond 0.100 mmolscale by adjusting the described volumes by the multiple of the scale. A“deprotection solution” was prepared by combining in a 40 mL glass vialtrifluoroacetic acid (22 mL), phenol (1.325 g), water (1.25 mL) andtriisopropylsilane (0.5 mL). The resin was removed from the reactionvessel and transferred to a 4 mL glass vial. To the vial was added the“deprotection solution” (2.0 mL). The mixture was vigorously mixed in ashaker (1000 RPM for 1 minute, then 500 RPM for 1-2 h). The mixture wasfiltered through a 0.2 micron syringe filter and the solids wereextracted with the “deprotection solution” (1.0 mL) or TFA (1.0 mL). Toa 24 mL test tube charged with the combined filtrates was added Et₂O (15mL). The mixture was vigorously mixed upon which a significant amount ofa white solid precipitated. The mixture was centrifuged for 5 minutes,then the solution was decanted away from the solids and discarded. Thesolids were suspended in Et₂O (20 mL); then the mixture was centrifugedfor 5 minutes; and the solution was decanted away from the solids anddiscarded. For a final time, the solids were suspended in Et₂O (20 mL);the mixture was centrifuged for 5 minutes; and the solution was decantedaway from the solids and discarded to afford the crude peptide as awhite to off-white solid.

Cyclization Method A:

All manipulations were performed manually unless noted. The procedure of“Cyclization Method A” describes an experiment performed on a 0.100 mmolscale, where the scale is determined by the amount of Rink linker boundto the resin that was used to generate the peptide. This scale is notbased on a direct determination of the quantity of peptide used in theprocedure. The procedure can be scaled beyond 0.100 mmol scale byadjusting the described volumes by the multiple of the scale.

The crude peptide solids were dissolved in MeCN:aq. 0.1M NH₄OAc (1:1) toa total volume of 18-22 mL, and the solution was carefully then adjustedto pH=8.5-9.0 using aq NaOH (1.0M). The solution was then allowed tostand without stirring for 12-18 h. The reaction solution wasconcentrated and the residue was then dissolved in DMSO:MeOH. Thissolution was subjected to reverse-phase HPLC purification to afford thedesired cyclic peptide.

General Synthetic Sequence A:

“General Synthetic Sequence A” describes a general sequence ofprocedures that were used to afford the cyclic peptides describedherein. For the purposes of this general procedure, the procedures of“Symphony Method A” are interchangeable with those of “Prelude MethodA”. To a 10 mL polypropylene solid-phase reaction vessel was added“Biotin Resin” (see below) (161 mg, 0.050 mmol), and the reaction vesselwas placed on the Prelude peptide synthesizer. For the followingprocedures, the same amounts of reagents described above for a 0.100mmol scale were used, although in this general synthetic sequence theamount of resin used corresponds to a 0.050 mmol scale. “Prelude MethodA: Resin-swelling procedure” was followed. Then a series of amino acidscouplings was sequentially performed on the Prelude following “PreludeMethod A: Single-coupling procedure” if the N-terminus of theresin-bound peptide was a primary amine or “Prelude Method A:Double-coupling procedure” if the N-terminus of the resin-bound peptidewas a secondary amine. “Prelude Method A: Chloroacetyl chloride couplingprocedure” was followed; then “Global Deprotection Method A” wasfollowed; then “Cyclization Method A” was followed.

General Synthetic Sequence B:

“General Synthetic Sequence B” describes a general sequence ofprocedures that were used to afford the cyclic peptides describedherein. For the purposes of this general procedure, the procedures of“Symphony Method A” are interchangeable with those of “Prelude MethodA”. To a 10 mL polypropylene solid-phase reaction vessel was addedRink-Merrifield resin (178 mg, 0.100 mmol), and the reaction vessel wasplaced on the Prelude peptide synthesizer. “Prelude Method A:Resin-swelling procedure” was followed. Then a series of amino acidscouplings was sequentially performed on the Prelude following “PreludeMethod A: Single-coupling procedure” if the N-terminus of theresin-bound peptide was a primary amine or “Prelude Method A:Double-coupling procedure” if the N-terminus of the resin-bound peptidewas a secondary amine. “Prelude Method A: Chloroacetyl chloride couplingprocedure” was followed; then “Global Deprotection Method A” wasfollowed; then “Cyclization Method A” was followed.

Preparation of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-ethoxypropanoicacid

Step 1

To a solution of (S)-2-amino-3-ethoxypropanoic acid (1.5 g, 11.3 mmol)in THF (38 ml) and water (19 ml) was added sodium bicarbonate (2.37 g,28.2 mmol) and Fmoc-OSu (3.80 g, 11.3 mmol). The resulting mixture wasstirred for 16 h. After removal of THF, the residue was acidified with 1N HCl, extracted with ethyl acetate, dried over Na₂SO₄, thenconcentrated to afford(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-ethoxypropanoic acidas a white solid, 3.6 g (90%).

Step 2

A mixture containing paraformaldehyde (1.825 g, 60.8 mmol),(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-ethoxypropanoic acid(3.6 g, 10.1 mmol), and p-toluenesulfonic acid (0.174 g, 1.01 mmol) intoluene (100 mL) was refluxed with Dean-Stark azeotropic removal ofwater for 2 h. The reaction was then cooled to RT, washed with aq. sat.sodium bicarbonate solution, followed by brine, dried over MgSO₄, thenfiltered and concentrated in vacuo to afford (S)-(9H-fluoren-9-yl)methyl4-(ethoxymethyl)-5-oxooxazolidine-3-carboxylate as a yellow oil, 5.16 g.

Step 3

(S)-(9H-fluoren-9-yl)methyl4-(2-methoxyethyl)-5-oxooxazolidine-3-carboxylate (0.4 g, 1.089 mmol)was dissolved in CHCl₃ (50 mL) and to the solution was addedtriethylsilane (0.869 mL, 5.44 mmol) followed by TFA (0.923 mL, 12.0mmol). The solution was stirred at RT under positive pressure of N₂ for18 h. The solution was then concentrated to afford an oil residue. Theresidue was dissolved in EtOAc and then extracted with aq. sat. sodiumbicarbonate (2×100 mL). The aqueous phase and all solids suspended atthe phase interface were collected. This mixture was acidified to pH 4-5using aq. HCl, upon which a precipitate formed. The mixture wasextracted with EtOAc (200 mL). The organic phase washed with brine,dried over MgSO₄, filtered and concentrated in vacuo to afford(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-methoxybutanoicacid as a white solid, 0.35 g (87%) yield).

Preparation of Example 5001

Example 5001 was prepared following “General Synthetic Sequence A”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 20-60% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 3.5 mg, and itsestimated purity by LCMS analysis was 100%. Analysis condition A:Retention time=1.68 min; ESI-MS(+) m/z 1171.8 (M+2H); Analysis conditionB: Retention time=2.83 min; ESI-MS(+) m/z 1171.7 (M+2H); ESI-HRMS(+)m/z: Calculated: 1170.5781; Found: 1170.5776.

Preparation of Example 5002

Example 5002 was prepared following “General Synthetic Sequence A”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 9.7 mg, and itsestimated purity by LCMS analysis was 100%. Analysis condition A:Retention time=1.42 min; ESI-MS(+) m/z 1128.8 (M+2H); Analysis conditionB: Retention time=2.57 min; ESI-MS(−) m/z 1126.8 (M−2H); ESI-HRMS(+)m/z: Calculated: 1128.5345 Found: 1128.5349.

Preparation of Example 5003

Example 5003 was prepared following “General Synthetic Sequence A”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: waters xbridge c-18, 19×200 mm, 5-μm particles;Mobile Phase A: 5:95 methanol:water with 0.1% trifluoroacetic acid;Mobile Phase B: 95:5 methanol:water with 0.1% trifluoroacetic acid;Gradient: 30-70% B over 30 minutes, then a 5-minute hold at 100% B;Flow: 20 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 11.0mg, and its estimated purity by LCMS analysis was 99%. Analysiscondition A: Retention time=1.44 min; ESI-MS(+) m/z 1172.4 (M+2H);Analysis condition B: Retention time=2.55 min; ESI-MS(+) m/z 1172.3(M+2H); ESI-HRMS(+) m/z: Calculated: 1171.0847; Found: 1171.0862.

Preparation of Example 5004

Example 5004 was prepared following “General Synthetic Sequence A”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: waters xbridge c-18, 19×200 mm, 5-μm particles;Mobile Phase A: 5:95 methanol:water with 0.1% trifluoroacetic acid;Mobile Phase B: 95:5 methanol:water with 0.1% trifluoroacetic acid;Gradient: 30-70% B over 30 minutes, then a 5-minute hold at 100% B;Flow: 20 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 8.9mg, and its estimated purity by LCMS analysis was 96%. Analysiscondition A: Retention time=1.52 min; ESI-MS(+) m/z 1151.2 (M+2H);Analysis condition B: Retention time=2.61 min; ESI-MS(+) m/z 1151.3(M+2H); ESI-HRMS(+) m/z: Calculated: 1150.6078; Found: 1150.6096.

Preparation of Example 5006

Example 5006 was prepared following “General Synthetic Sequence B”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 30-70% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 26.3 mg, and its estimatedpurity by LCMS analysis was 99%. Analysis condition A: Retentiontime=1.32 min; ESI-MS(+) m/z 1078.3 (M+2H); Analysis condition B:Retention time=2.42 min; ESI-MS(+) m/z 1078.2 (M+2H); ESI-HRMS(+) m/z:Calculated: 1077.5387 Found: 1077.5396.

Preparation of Example 5007

Example 5007 was prepared following “General Synthetic Sequence B”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 30-70% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 36.9 mg, and its estimatedpurity by LCMS analysis was 100%; Analysis condition A: Retentiontime=1.10 min; ESI-MS(+) m/z 923.7 (M+2H); Analysis condition B:Retention time=2.29 min; ESI-MS(+) m/z 923.7 (M+2H).

Preparation of Example 5008

Example 5008 was prepared following “General Synthetic Sequence B” on a0.600 mmol scale. The crude material was purified via preparative LC/MSwith the following conditions: Column: waters xbridge c-18, 19×200 mm,5-μm particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammoniumacetate; Gradient: 25-65% B over 30 minutes, then a 5-minute hold at100% B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The material was furtherpurified via preparative LC/MS with the following conditions: Column:XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95methanol:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5methanol:water with 0.1% trifluoroacetic acid; Gradient: 25-70% B over30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 49.9 mg, and its estimatedpurity by LCMS analysis was 99%; Analysis condition A: Retentiontime=1.15 min; ESI-MS(+) m/z 1064.9 (M+2H); Analysis condition B:Retention time=2.19 min; ESI-MS(+) m/z 1064.2 (M+2H); ESI-HRMS(+) m/z:Calculated: 1063.5231; Found: 1063.5222.

Preparation of Example 5009

Example 5009 was prepared following “General Synthetic Sequence B”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 55-95% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 9.0 mg, and its estimatedpurity by LCMS analysis was 99%; Analysis condition A: Retentiontime=1.60 min; ESI-MS(+) m/z 1057.4 (M+2H); Analysis condition B:Retention time=2.81 min; ESI-MS(+) m/z 1056.6 (M+2H); ESI-HRMS(+) m/z:Calculated: 1056.0087; Found: 1056.0069.

Preparation of Example 5010

Example 5010 was prepared following “General Synthetic Sequence B”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 14.9 mg, and its estimatedpurity by LCMS analysis was 96%; Analysis condition A: Retentiontime=1.58 min; ESI-MS(+) m/z 1098.7 (M+2H); Analysis condition B:Retention time=2.76 min; ESI-MS(−) m/z 1096.6 (M−2H); ESI-HRMS(+) m/z:Calculated: 1098.0557; Found: 1098.0554.

Preparation of Example 5011

Example 5011 was prepared following “General Synthetic Sequence B”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 50-90% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 25.8 mg, and its estimatedpurity by LCMS analysis was 100%. Analysis condition A: Retentiontime=1.58 min; ESI-MS(+) m/z 1136.7 (M+2H); Analysis condition B:Retention time=2.75 min; ESI-MS(−) m/z 1134.3 (M−2H); ESI-HRMS(+) m/z:Calculated: 1135.5535; Found: 1135.5528.

Preparation of Example 5012

Example 5012 was prepared following “General Synthetic Sequence B”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 50-95% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 13.8 mg, and its estimatedpurity by LCMS analysis was 100%; Analysis condition A: Retentiontime=1.59 min; ESI-MS(−) m/z 1140.3 (M−2H); Analysis condition B:Retention time=2.81 min; ESI-MS(+) m/z 1142.4 (M+2H; ESI-HRMS(+) m/z:Calculated: 1141.5535 Found: 1141.5539.

Preparation of Example 5013

Example 5013 was prepared following “General Synthetic Sequence B”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 40-80% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 12.1 mg, and its estimatedpurity by LCMS analysis was 100%; Analysis condition A: Retentiontime=1.57 min; ESI-MS(−) m/z 1075.8 (M−2H; Analysis condition B:Retention time=2.75 min; ESI-MS(+) m/z 1077.8 (M+2H); ESI-HRMS(+) m/z:Calculated: 1077.0322 Found: 1077.0330.

Preparation of Example 5014

Example 5014 was prepared following “General Synthetic Sequence B”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 35-80% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 15.7 mg, and its estimatedpurity by LCMS analysis was 100%; Analysis condition A: Retentiontime=1.36 min; ESI-MS(+) m/z 1035.8 (M+2H); Analysis condition B:Retention time=2.52 min; ESI-MS(−) m/z 1033.7 (M−2H); ESI-HRMS(+) m/z:Calculated: 1034.9885; Found: 1034.9887.

Preparation of Example 5015

Example 5015 was prepared as follows: To a 1 dram vial charged withExample 5006 (5.8 mg, 2.7 μmol) was added dry NMP. The mixture wasagitated until a homogeneous solution formed. To the solution was addedDIPEA (0.025 mL, 0.143 mmol), then 2,5-dioxopyrrolidin-1-yl2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oate (3.5mg, 5.1 μmol). The vial was placed on a shaker rotating at 500 rpm for30 minutes. The reaction was quenched via the addition of ethanolamine(0.020 mL). The crude material was purified via preparative LC/MS withthe following conditions: Column: XBridge C18, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at100% B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 2.0 mg, and its estimated purity by LCMS analysis was 99%. Analysiscondition A: Retention time=1.52 min; ESI-MS(−) m/z 1361.4 (M−2H);Analysis condition B: Retention time=2.61 min; ESI-MS(−) m/z 1361.7(M−2H).

Preparation of Example 5016

Example 5016 was prepared as follows: To a 1 dram vial charged withExample 5006 (5.8 mg, 2.7 μmol) was added dry NMP. The mixture wasagitated until a homogeneous solution formed. To the solution was addedDIPEA (0.025 mL, 0.143 mmol), then 2,5-dioxopyrrolidin-1-yl2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatetraheptacontan-74-oate(3.27 mg, 2.69 μmol). The vial was placed on a shaker rotating at 500rpm for 40 minutes. The reaction was quenched via the addition ofethanolamine (0.020 mL). The crude material was purified via preparativeLC/MS with the following conditions: Column: XBridge C18, 19×200 mm,5-μm particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammoniumacetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at100% B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 2.6 mg, and its estimated purity by LCMS analysis was 100%. Analysiscondition A: Retention time=1.55 min; ESI-MS(−) m/z 1625.4 (M−2H);Analysis condition B: Retention time=2.67 min; ESI-MS(−) m/z 1625.7(M−2H).

Preparation of Example 5017

Example 5017 was prepared as follows: To a 1 dram vial charged withExample 5006 (5.8 mg, 2.7 μmol) was added dry NMP. The mixture wasagitated until a homogeneous solution formed. To the solution was addedDIPEA (0.025 mL, 0.143 mmol), then 2,5-dioxopyrrolidin-1-yl2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,134,137,140,143,146-nonatetracontaoxanonatetracontahectan-149-oate(6.2 mg, 2.7 μmol). The vial was placed a shaker rotating at 500 rpm for30 minutes. The reaction was quenched via the addition of ethanolamine(0.020 mL). The crude material was purified via preparative LC/MS withthe following conditions: Column: XBridge C18, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 methanol:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammoniumacetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at100% B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 3.7 mg, and its estimated purity by LCMS analysis was 100%; Analysiscondition A: Retention time=1.63 min; ESI-MS(+) m/z 882.5 (M+5H;Analysis condition B: Retention time=2.74 min; ESI-MS(+) m/z 882.6(M+5H).

Preparation of Example 5018

Example 5018 was prepared as follows: To a 1 dram vial charged withExample 5006 (5.8 mg, 2.7 μmol) was added dry NMP. The mixture wasagitated until a homogeneous solution formed. To the solution was addedDIPEA (0.025 mL, 0.143 mmol), then 2,5-dioxopyrrolidin-1-yl2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110-heptatriacontaoxatridecahectan-113-oate(4.8 mg, 2.7 μmol). The vial was placed a shaker rotating at 500 rpm for40 minutes. The reaction was quenched via the addition of ethanolamine(0.020 mL). The crude material was purified via preparative LC/MS withthe following conditions: Column: XBridge C18, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 methanol:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammoniumacetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at100% B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 3.0 mg, and its estimated purity by LCMS analysis was 100%; Analysiscondition A: Retention time=1.63 min; ESI-MS(−) m/z 1914.0 (M−2H);Analysis condition B: Retention time=2.70 min; ESI-MS(−) m/z 1912.7(M−2H).

Analytical Data:

Mass Spectrometry: “ESI-MS(+)” signifies electrospray ionization massspectrometry performed in positive ion mode; “ESI-MS(−)” signifieselectrospray ionization mass spectrometry performed in negative ionmode; “ESI-HRMS(+)” signifies high-resolution electrospray ionizationmass spectrometry performed in positive ion mode; “ESI-HRMS(−)”signifies high-resolution electrospray ionization mass spectrometryperformed in negative ion mode. The detected masses are reportedfollowing the “m/z” unit designation. Compounds with exact massesgreater than 1000 were often detected as double-charged ortriple-charged ions.

General Procedures: Peptide Synthesis

The macrocyclic peptides of the present disclosure can be produced bymethods known in the art, such as they can be synthesized chemically,recombinantly in a cell free system, recombinantly within a cell or canbe isolated from a biological source. Chemical synthesis of amacrocyclic peptide of the present disclosure can be carried out using avariety of art recognized methods, including stepwise solid phasesynthesis, semi-synthesis through the conformationally-assistedre-ligation of peptide fragments, enzymatic ligation of cloned orsynthetic peptide segments, and chemical ligation. A preferred method tosynthesize the macrocyclic peptides and analogs thereof described hereinis chemical synthesis using various solid-phase techniques such as thosedescribed in Chan, W. C. et al., eds., Fmoc Solid Phase Synthesis,Oxford University Press, Oxford (2000); Barany, G. et al., The Peptides:Analysis, Synthesis, Biology, Vol. 2: “Special Methods in PeptideSynthesis, Part A”, pp. 3-284, Gross, E. et al., eds., Academic Press,New York (1980); and in Stewart, J. M. et al., Solid-Phase PeptideSynthesis, 2nd Edition, Pierce Chemical Co., Rockford, Ill. (1984). Thepreferred strategy is based on the Fmoc (9-Fluorenylmethylmethyl-oxycarbonyl) group for temporary protection of the α-amino group,in combination with the tert-butyl group for temporary protection of theamino acid side chains (see for example Atherton, E. et al., “TheFluorenylmethoxycarbonyl Amino Protecting Group”, in The Peptides:Analysis, Synthesis, Biology, Vol. 9: “Special Methods in PeptideSynthesis, Part C”, pp. 1-38, Undenfriend, S. et al., eds., AcademicPress, San Diego (1987).

The peptides can be synthesized in a stepwise manner on an insolublepolymer support (also referred to as “resin”) starting from theC-terminus of the peptide. A synthesis is begun by appending theC-terminal amino acid of the peptide to the resin through formation ofan amide or ester linkage. This allows the eventual release of theresulting peptide as a C-terminal amide or carboxylic acid,respectively.

The C-terminal amino acid and all other amino acids used in thesynthesis are required to have their α-amino groups and side chainfunctionalities (if present) differentially protected such that theα-amino protecting group may be selectively removed during thesynthesis. The coupling of an amino acid is performed by activation ofits carboxyl group as an active ester and reaction thereof with theunblocked α-amino group of the N-terminal amino acid appended to theresin. The sequence of α-amino group deprotection and coupling isrepeated until the entire peptide sequence is assembled. The peptide isthen released from the resin with concomitant deprotection of the sidechain functionalities, usually in the presence of appropriate scavengersto limit side reactions. The resulting peptide is finally purified byreverse phase HPLC.

The synthesis of the peptidyl-resins required as precursors to the finalpeptides utilizes commercially available cross-linked polystyrenepolymer resins (Novabiochem, San Diego, Calif.; Applied Biosystems,Foster City, Calif.). Preferred solid supports are:4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methylbenzhydrylamine resin (Rink amide MBHA resin);9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin (Sieber amide resin);4-(9-Fmoc)aminomethyl-3,5-dimethoxyphenoxy)valeryl-aminomethyl-Merrifieldresin (PAL resin), for C-terminal carboxamides. Coupling of first andsubsequent amino acids can be accomplished using HOBt, 6-Cl-HOBt or HOAtactive esters produced from DIC/HOBt, HBTU/HOBt, BOP, PyBOP, or fromDIC/6-Cl-HOBt, HCTU, DIC/HOAt or HATU, respectively. Preferred solidsupports are: 2-Chlorotrityl chloride resin and9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin (Sieber amide resin) forprotected peptide fragments. Loading of the first amino acid onto the2-chlorotrityl chloride resin is best achieved by reacting theFmoc-protected amino acid with the resin in dichloromethane and DIEA. Ifnecessary, a small amount of DMF may be added to facilitate dissolutionof the amino acid.

The syntheses of the peptide analogs described herein can be carried outby using a single or multi-channel peptide synthesizer, such as an CEMLiberty Microwave synthesizer, or a Protein Technologies, Inc. Prelude(6 channels) or Symphony (12 channels) synthesizer.

Useful Fmoc amino acids derivatives are shown below.

Examples of Orthogonally Protected Amino Acids used in Solid PhaseSynthesis

The peptidyl-resin precursors for their respective peptides may becleaved and deprotected using any standard procedure (see, for example,King, D. S. et al., Int. J. Peptide Protein Res., 36:255-266 (1990)). Adesired method is the use of TFA in the presence of water and TIS asscavengers. Typically, the peptidyl-resin is stirred in TFA/water/TIS(94:3:3, v:v:v; 1 mL/100 mg of peptidyl resin) for 2-6 hrs at roomtemperature. The spent resin is then filtered off and the TFA solutionis concentrated or dried under reduced pressure. The resulting crudepeptide is either precipitated and washed with Et₂O or is redissolveddirectly into DMSO or 50% aqueous acetic acid for purification bypreparative HPLC.

Peptides with the desired purity can be obtained by purification usingpreparative HPLC, for example, on a Waters Model 4000 or a ShimadzuModel LC-8A liquid chromatograph. The solution of crude peptide isinjected into a YMC S5 ODS (20×100 mm) column and eluted with a lineargradient of MeCN in water, both buffered with 0.1% TFA, using a flowrate of 14-20 mL/min with effluent monitoring by UV absorbance at 220nm. The structures of the purified peptides can be confirmed byelectro-spray MS analysis.

List of non-naturally occurring amino acids referred to herein isprovided below.

The following abbreviations are employed in the Examples and elsewhereherein:

Ph=phenyl

Bn=benzyl

i-Bu=iso-butyl

i-Pr=iso-propyl

Me=methyl

Et=ethyl

Pr=n-propyl

Bu=n-butyl

t-Bu=tert-butyl

Trt=trityl

TMS=trimethylsilyl

TIS=triisopropylsilane

Et₂O=diethyl ether

HOAc or AcOH=acetic acid

MeCN or AcCN=acetonitrile

DMF=N,N-dimethylformamide

EtOAc=ethyl acetate

THF=tetrahydrofuran

TFA=trifluoroacetic acid

TFE=α,α,α-trifluoroethanol

Et₂NH=diethylamine

NMM=N-methylmorpholine

NMP=N-methylpyrrolidone

DCM=dichloromethane

TEA=triethylamine

min.=minute(s)

h or hr=hour(s)

L=liter

mL or ml=milliliter

μL=microliter

g=gram(s)

mg=milligram(s)

mol=mole(s)

mmol=millimole(s)

meq=milliequivalent

rt or RT=room temperature

sat or sat'd=saturated

aq.=aqueous

mp=melting point

BOP reagent=benzotriazol-1-yloxy-tris-dimethylamino-phosphoniumhexafluorophosphate (Castro's reagent)

PyBOP reagent=benzotriazol-1-yloxy-tripyrrolidino phosphoniumhexafluorophosphate

HBTU=2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate

HATU=O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate

HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate

T3P=2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide

DMAP=4-(dimethylamino)pyridine

DIEA=diisopropylethylamine

Fmoc or FMOC=fluorenylmethyloxycarbonyl

Boc or BOC=tert-butyloxycarbonyl

HOBT or HOBT•H₂O=1-hydroxybenzotriazole hydrate

Cl-HOBt=6-Chloro-benzotriazole

HOAT=1-hydroxy-7-azabenzotriazole

HPLC=high performance liquid chromatography

LC/MS=high performance liquid chromatography/mass spectrometry

MS or Mass Spec=mass spectrometry

NMR=nuclear magnetic resonance

Sc or SC=sub-cutaneous

IP or ip=intra-peritoneal

EXAMPLES Example 0001—Solid Phase Peptide Synthesis and Cyclization ofPeptides

The procedures described in this example, either in whole or in partwhere noted, were used to synthesize the macrocyclic peptides shown inTables 1, 2, 3, 4 and 5.

General protocol for solid-phase peptide synthesis and macrocyclization.On a Symphony Peptide Synthesizer (Protein Technology Inc. Tucson,Ariz.), Prelude Peptide Synthesizer (Protein Technology Inc. Tucson,Ariz.), or Liberty (CEM Matthews, N.C.), Sieber Amide resin (0.71mmol/g, 0.100 mmol, 141 mg) was swelled with DMF (7 mL×4 min) and mixedwith a gentle stream of N₂ every 30 seconds. The solvent was drained andthe following method was used to couple the first amino acid: the Fmocgroup was removed from the resin-supported building block by washing theresin twice with a solution of 20% piperidine in DMF (5 mL and 2.5minutes per wash) and mixing with a gentle stream of N₂ every 30seconds. The resin washed three times with DMF (5-8 mL and 1.5 min perwash). 2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetic acid (0.2 Msolution in DMF, 0.5 mmol) was then added, followed by couplingactivator (i.e., HATU (Chem-Impex Int'l, 0.4M solution in DMF, 1.25 mL,0.5 mmol)) and base (i.e., N-methyl morpholine (Aldrich, 0.8 M in DMF,1.25 mL, 1 mmol)). The reaction mixture was agitated by a gentle streamof nitrogen for 1 h. The reagents were drained from the reaction vessel,and the resin washed three times with DMF (5 mL×1.5 min). It should benoted that the typical reagents for the Liberty CEM were the following:HCTU (0.45 M in DMF) as the coupling activator, DIEA (2M in NMP) as thebase, and 5% piperazine in DMF with 0.1 M HOBt as the deprotectsolution.

The resulting resin-supported Fmoc-protected dipeptide was thensequentially deprotected and coupled with third amino acid and so forthin an iterative fashion to give the desired resin-supported product.

LCMS analysis was performed on a peptide aliquot, which was cleaved fromthe resin (analytical amount was treated with a TFA/TIS (96:4) solution(0.2 mL) at room temperature. Following confirmation of the desiredlinear sequence, the Fmoc group was removed from the N-terminus uponwashing the resin twice with a solution of 20% piperidine in DMF (5 mLand 2.5 minutes per wash) and vortexing the slurry. The resin washedwith DMF (2×5 mL). To the peptide-resin was added in succession2-chloroacetic acid (0.6 mmol, 57 mg), DMF (5.26 mL), and DIC (0.6 mmol,93 μL). The new slurry was vortexed for 1-2 days as which point thepeptide-resin washed with DMF (1×5 mL×1 min) and DCM (3×DCM×1 min).

The peptide was deprotected and cleaved from the resin upon treatmentwith a TFA/TIS (96:4) solution (10 mL) for 1 h. The resin was removed byfiltration, washed with cleavage cocktail (2×1 mL), the combinedfiltrates were added to Et₂O (10-15 mL) and the solution was chilled at0° C. in order to effect the peptide to precipitate out of solution. Theslurry is centrifuged to pellet the solids and the supernatant wasdecanted. Fresh Et₂O (25 mL) was added and the process was repeatedthree times to wash the solids. To the wet solids was added a solutionof 0.1 M NH₄HCO₃/Acetonitrile (from 1/1 to 3/1 (v/v), pH=8.6) or 6 Mguanidine HCl in 100 mM NaH₂PO₄ (pH=8.4). The solution was stirred for1-2 days and monitored by LCMS. The reaction solution was purified bypreparative HPLC to obtain the desired product.

General Analytical Protocols and Synthesis Methods Analytical Data:

Mass Spectrometry: “ESI-MS(+)” signifies electrospray ionization massspectrometry performed in positive ion mode; “ESI-MS(−)” signifieselectrospray ionization mass spectrometry performed in negative ionmode; “ESI-HRMS(+)” signifies high-resolution electrospray ionizationmass spectrometry performed in positive ion mode; “ESI-HRMS(−)”signifies high-resolution electrospray ionization mass spectrometryperformed in negative ion mode. The detected masses are reportedfollowing the “m/z” unit designation. Compounds with exact massesgreater than 1000 were often detected as double-charged ortriple-charged ions.

General Procedures: Symphony X Method A:

All manipulations were performed under automation on a Symphony Xpeptide synthesizer (Protein Technologies). All procedures wereperformed in a 10 mL polypropylene tube fitted with a bottom fit. Thetube connects to a the Symphony X peptide synthesizer through both thebottom and the top of the tube. DMF and DCM can be added through the topof the tube, which washes down the sides of the tube equally. Theremaining reagents are added through the bottom of the tube and pass upthrough the frit to contact the resin. All solutions are removed throughthe bottom of the tube. “Periodic agitation” describes a brief pulse ofN₂ gas through the bottom frit; the pulse lasts approximately 5 secondsand occurs every 30 seconds. Chloroacetyl chloride solutions in DMF wereused within 24 h of preparation. Amino acid solutions were generally notused beyond three weeks from preparation. HATU solutions were usedwithin 5 days of preparation. DMF=dimethylformamide;HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate; DIPEA=diisopropylethylamine;Rink=(2,4-dimethoxyphenyl)(4-alkoxyphenyl)methanamine, where “4-alkoxy”describes the position and type of connectivity to the polystyreneresin. The resin used is Merrifield polymer (polystyrene) with a Rinklinker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.56 mmol/gloading. Common amino acids used are listed below with side-chainprotecting groups indicated inside parenthesis.

Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH;Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH;Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH;Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH;Fmoc-[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH;Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH;Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

The procedures of “Symphony X Method A” describe an experiment performedon a 0.100 mmol scale, where the scale is determined by the amount ofRink linker bound to the resin. This scale corresponds to approximately178 mg of the Rink-Merrifield resin described above. All procedures canbe scaled beyond 0.100 mmol scale by adjusting the described volumes bythe multiple of the scale. Prior to amino acid coupling, all peptidesynthesis sequences began with a resin-swelling procedure, describedbelow as “Resin-swelling procedure”. Coupling of amino acids to aprimary amine N-terminus used the “Single-coupling procedure” describedbelow. Coupling of amino acids to a secondary amine N-terminus used the“Double-coupling procedure” described below. Coupling ofchloroacetylchloride to the N-terminus of the peptide is described bythe “Chloroacetyl chloride coupling procedure” detailed below.

Resin-Swelling Procedure A:

To a 10 mL polypropylene solid-phase reaction vessel was addedMerrifield:Rink resin (178 mg, 0.100 mmol). The resin washed (swelled)three times as follows: to the reaction vessel was added DMF (2.0 mL),upon which the mixture was periodically agitated for 10 minutes beforethe solvent was drained through the frit.

Single-Coupling Procedure A:

To the reaction vessel containing resin from the previous step was addedpiperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0mL). The mixture was periodically agitated for 3 minutes and then thesolution was drained through the frit. The resin washed successively sixtimes as follows: for each wash, DMF (2.0 mL) was added through the topof the vessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added the amino acid (0.2M in DMF, 1.0 mL, 2 eq),then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA (0.4M in DMF,1.0 mL, 4 eq). The mixture was periodically agitated for 15 minutes,then the reaction solution was drained through the frit. The resinwashed successively four times as follows: for each wash, DMF (2.0 mL)was added through the top of the vessel and the resulting mixture wasperiodically agitated for 30 seconds before the solution was drainedthrough the frit. To the reaction vessel was added acetic anhydride (2.0mL). The mixture was periodically agitated for 10 minutes, then thesolution was drained through the frit. The resin washed successivelyfour times as follows: for each wash, DMF (2.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 90 seconds before the solution was drained through the frit. Theresulting resin was used directly in the next step.

Double-Coupling Procedure A:

To the reaction vessel containing resin from the previous step was addedpiperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0mL). The mixture was periodically agitated for 3 minutes and then thesolution was drained through the frit. The resin washed successively sixtimes as follows: for each wash, DMF (2.0 mL) was added through the topof the vessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added the amino acid (0.2M in DMF, 1.0 mL, 2 eq),then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA (0.4M in DMF,1.0 mL, 4 eq). The mixture was periodically agitated for 15 minutes,then the reaction solution was drained through the frit. The resin wastwice washed as follows: for each wash, DMF (2.0 mL) was added throughthe top of the vessel and the resulting mixture was periodicallyagitated for 30 seconds before the solution was drained through thefrit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.0mL, 2 eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA(0.4M in DMF, 1.0 mL, 4 eq). The mixture was periodically agitated for15 minutes, then the reaction solution was drained through the frit. Theresin was twice washed as follows: for each wash, DMF (2.0 mL) was addedthrough the top of the vessel and the resulting mixture was periodicallyagitated for 90 seconds before the solution was drained through thefrit. To the reaction vessel was added acetic anhydride (2.0 mL). Themixture was periodically agitated for 10 minutes, then the solution wasdrained through the frit. The resin washed successively four times asfollows: for each wash, DMF (2.0 mL) was added through the top of thevessel and the resulting mixture was periodically agitated for 90seconds before the solution was drained through the frit. The resultingresin was used directly in the next step.

Symphony Amino Acid N-Terminal Stop Procedure:

To a 10 mL polypropylene solid-phase reaction vessel was addedMerrifield:Rink resin (178 mg, 0.100 mmol). The resin washed (swelled)three times as follows: to the reaction vessel was added DMF (2.0 mL),upon which the mixture was periodically agitated for 10 minutes beforethe solvent was drained through the frit.

To the reaction vessel containing Rink resin from the previous step wasadded piperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0mL). The mixture was periodically agitated for 3 minutes and then thesolution was drained through the frit. The resin washed successively sixtimes as follows: for each wash, DMF (2.0 mL) was added through the topof the vessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added the amino acid (0.2M in DMF, 1.0 mL, 2 eq),then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA (0.4M in DMF,1.0 mL, 4 eq). The mixture was periodically agitated for 15 minutes,then the reaction solution was drained through the frit. The resinwashed successively four times as follows: for each wash, DMF (2.0 mL)was added through the top of the vessel and the resulting mixture wasperiodically agitated for 30 seconds before the solution was drainedthrough the frit. To the reaction vessel was added acetic anhydride (2.0mL). The mixture was periodically agitated for 10 minutes, then thesolution was drained through the frit. The resin washed successivelyfour times as follows: for each wash, DMF (2.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 90 seconds before the solution was drained through the frit. To thereaction vessel was added piperidine:DMF (20:80 v/v, 2.0 mL). Themixture was periodically agitated for 3 minutes and then the solutionwas drained through the frit. To the reaction vessel was addedpiperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. The resin washed successively five times as follows: for eachwash, DMF (2.0 mL) was added through the top of the vessel and theresulting mixture was periodically agitated for 90 seconds before thesolution was drained through the frit. The resin washed successivelythree times as follows: for each wash, DCM (2.0 mL) was added throughthe top of the vessel and the resulting mixture was periodicallyagitated for 90 seconds before the solution was drained through thefrit. The resulting resin was placed under a stream on nitrogen for 15minutes.

Chloroacetyl Chloride Coupling Procedure A:

To the reaction vessel containing the resin from the previous step wasadded piperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0mL). The mixture was periodically agitated for 3 minutes and then thesolution was drained through the frit. The resin washed successively sixtimes as follows: for each wash, DMF (2.0 mL) was added through the topof the vessel and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added DIPEA (0.4M in DMF, 4.0 mL, 16 eq), thenchloroacetyl chloride (0.8M in DMF, 1.50 mL). The mixture wasperiodically agitated for 30 minutes, then the solution was drainedthrough the frit. The resin washed successively three times as follows:for each wash, DMF (2.0 mL) was added to top of the vessel and theresulting mixture was periodically agitated for 90 seconds before thesolution was drained through the frit. The resin washed successivelyfour times as follows: for each wash, CH₂Cl₂ (2.0 mL) was added to topof the vessel and the resulting mixture was periodically agitated for 90seconds before the solution was drained through the frit. The resultingresin was placed under a N₂ stream for 15 minutes.

Chloroacetic Acid Coupling Procedure A:

To the reaction vessel containing resin from the previous step was addedpiperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodicallyagitated for 3 minutes and then the solution was drained through thefrit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0mL). The mixture was periodically agitated for 3 minutes and then thesolution was drained through the frit. The resin washed successivelyfour times as follows: for each wash, DMF (2.0 mL) was added through thetop of the vessel and the resulting mixture was periodically agitatedfor 30 seconds before the solution was drained through the frit. To thereaction vessel was added the chloroacetic acid (0.2M in DMF, 1.0 mL, 2eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA (0.4M inDMF, 1.0 mL, 4 eq). The mixture was periodically agitated for 15minutes, then the reaction solution was drained through the frit. Theresin washed successively three times as follows: for each wash, DMF(2.0 mL) was added through the top of the vessel and the resultingmixture was periodically agitated for 30 seconds before the solution wasdrained through the frit. The resin washed successively four times asfollows: for each wash, CH₂Cl₂ (2.0 mL) was added through the top of thevessel and the resulting mixture was periodically agitated for 90seconds before the solution was drained through the frit. The resultingresin was dried for 5 minutes.

Global Deprotection Method A:

All manipulations were performed manually unless noted. The procedure of“Global Deprotection Method A” describes an experiment performed on a0.100 mmol scale, where the scale is determined by the amount of Rinklinker bound to the resin. The procedure can be scaled beyond 0.100 mmolscale by adjusting the described volumes by the multiple of the scale. A“deprotection solution” was prepared by combining in a 40 mL glass vialtrifluoroacetic acid (22 mL), phenol (1.325 g), water (1.25 mL) andtriisopropylsilane (0.5 mL). The resin was removed from the reactionvessel and transferred to a 4 mL glass vial. To the vial was added the“deprotection solution” (2.0 mL). The mixture was vigorously mixed in ashaker (1000 RPM for 1 minute, then 500 RPM for 90 minutes). The mixturewas filtered through 10 mL polypropylene tube fitted with a bottom fritallowing for dropwise addition to a 24 mL test tube containing 15 mL ofdiethyl ether resulting a white precipitate. The solids (resin) in thetube were extracted once with the “deprotection solution” (1.0 mL)allowing dropwise addition to the ether. The mixture was centrifuged for7 minutes, then the solution was decanted away from the solids anddiscarded. The solids were suspended in Et₂O (20 mL); then the mixturewas centrifuged for 5 minutes; and the solution was decanted away fromthe solids and discarded. For a final time, the solids were suspended inEt₂O (20 mL); the mixture was centrifuged for 5 minutes; and thesolution was decanted away from the solids and discarded to afford thecrude peptide as a white to off-white solid.

Cyclization Method A:

All manipulations were performed manually unless noted. The procedure of“Cyclization Method A” describes an experiment performed on a 0.100 mmolscale, where the scale is determined by the amount of Rink linker boundto the resin that was used to generate the peptide. This scale is notbased on a direct determination of the quantity of peptide used in theprocedure. The procedure can be scaled beyond 0.100 mmol scale byadjusting the described volumes by the multiple of the scale. The crudepeptide solids were dissolved in Methanol (10 mL), and the solution wasthen carefully adjusted to pH=9.0-11 using N,N-Diisopropylamine. Thesolution was then allowed to stir for 18-24 h. The reaction solution wasconcentrated and the residue was then dissolved in MeOH. This solutionwas subjected to reverse-phase HPLC purification to afford the desiredcyclic peptide.

Analysis Condition A:

Column: X-Bridge C18, 2.0×50 mm, 3.5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10 mM ammonium acetate; Temperature: 40° C.;Gradient: 0% B, 0-100% B over 8 minutes, then a 1.0-minute hold at 100%B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

Preparation of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-undecanamidohexanoicacid

To a round-bottom flask charged with(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoic acid(2.5 g, 6.79 mmol), undecanoyl chloride (1.647 ml, 7.46 mmol), anddichloromethane (27 ml) was added N-ethyl-N-isopropylpropan-2-amine(3.56 ml, 20.36 mmol). The initial suspension immediately turns yellowand then clear. After 10 minutes a solid begins to precipitate. Thereaction was stirred for 20 hours at room temperature. The reactionmixture was diluted with 20 ml dichloromethane and poured into saturatedammonium chloride solution. The layers were separated and the aqueouswas washed with a 20% methanol/chloroform solution. The combinedorganics were washed with brine, dried over magnesium sulfate, filtered,and concentrated to give a sticky yellow solid. The resulting residuewas subjected to silica gel chromatography (0-5%methanol/dichloromethane gradient) to afford(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-undecanamidohexanoicacid (2.81 g, 5.24 mmol, 77% yield) as a yellow foam. ¹H NMR (400 MHz,CHLOROFORM-d) δ 7.80-7.71 (m, 2H), 7.63-7.52 (m, 2H), 7.39 (t, J=7.0 Hz,2H), 7.34-7.27 (m, 2H), 4.49-4.29 (m, 2H), 4.27-4.14 (m, 1H), 3.24 (br.s., 1H), 2.35 (t, J=7.5 Hz, 1H), 2.29-2.06 (m, 2H), 1.89 (br. s., 1H),1.86-1.72 (m, 1H), 1.72-1.63 (m, 1H), 1.62-1.46 (m, 4H), 1.39-1.14 (m,18H), 0.92-0.86 (m, 3H).

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-tetradecanamidohexanoicacid

¹H NMR (400 MHz, DMSO-d₆) δ 7.88 (dd, J=7.2, 4.1 Hz, 2H), 7.77-7.69 (m,2H), 7.44-7.37 (m, 2H), 7.35-7.28 (m, 2H), 4.29-4.17 (m, 2H), 3.95-3.84(m, 1H), 3.63-3.52 (m, 1H), 3.10 (q, J=7.4 Hz, 1H), 3.05-2.94 (m, 2H),2.01 (t, J=7.4 Hz, 2H), 1.46 (d, J=6.8 Hz, 3H), 1.34 (dd, J=13.1, 5.0Hz, 3H), 1.24-1.20 (m, 22H), 0.86-0.82 (m, 3H)

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-stearamidohexanoicacid

¹H NMR (400 MHz, DMSO-d₆) δ 7.92-7.84 (m, 2H), 7.72 (d, J=7.5 Hz, 2H),7.44-7.36 (m, 2H), 7.36-7.26 (m, 2H), 4.35-4.22 (m, 2H), 4.20 (d, J=7.5Hz, 1H), 3.94-3.85 (m, 1H), 3.00 (br. s., 2H), 2.17 (t, J=7.4 Hz, 1H),2.09-1.91 (m, 1H), 1.69 (d, J=6.8 Hz, 1H), 1.65-1.51 (m, 1H), 1.46 (d,J=7.0 Hz, 2H), 1.34 (dd, J=13.1, 5.0 Hz, 2H), 1.24-1.19 (m, 30H),0.87-0.82 (m, 3H).

Preparation of Modified Rink Resin A

A 20 ml scintillation vial was charged with Merrifield Rink resin (0.56mmol/g loading) (1.0 g, 0.560 mmol). The resin was swelled in 5 ml DMFfor 10 minutes. A solution of 8 ml of a 20:80 piperidine:DMF solutionwas added and the resulting suspension was shaken on the mini-shaker for2 hours. The resin was isolated by transferring the contents of the vialinto 10 ml polypropylene reaction tube and filtering by vacuumfiltration. The resin washed with 30 ml DMF followed by 30 mldichloromethane and lastly with 5 ml diethyl ether. The resin wastransferred to 20 ml vial. To the vial containing the resin was added 5ml DMF to swell the resin. After 10 minutes(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-undecanamidohexanoicacid (0.601 g, 1.120 mmol), 0.2M2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphate(V) in DMF (5.60 ml, 1.120 mmol), and 0.4MN-ethyl-N-isopropylpropan-2-amine in DMF (5.60 ml, 2.240 mmol) wereadded. The vial was shaken overnight on the mini-shaker. The resin wasisolated by transferring the contents of the vial into 10 mlpolypropylene reaction tube and filtering by vacuum filtration. Theresin washed with 50 ml DMF, 50 ml dichloromethane, and 10 ml diethylether. The resulting resin was dried in vacuo and used as a 0.56 mmol/gloading.

Preparation of Modified Rink Resin B

Modified rink resin B was made following identical procedure to ModifiedRink resin A.

Preparation of Modified Rink Resin C

Modified rink resin C was made following identical procedure to ModifiedRink resin A with the only exception being the N-Fmoc-N Palmitoyl-LLysine was purchased from Chem-Impex International.

Preparation of Modified Rink Resin D

Modified rink resin D was made following identical procedure to ModifiedRink resin A.

Preparation of Modified 2-Chlorotrityl Chloride Resin A

To a 40 mL vial was added 2-chlorotrityl chloride resin (1.42 mmol/gloading) (1.985 g, 2.78 mmol). The resin was swelled in 15 mldichloromethane for 10 minutes. A solution of (0.5 g, 0.869 mmol),FMOC-21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid in 2 mldichloromethane followed by N-ethyl-N-isopropylpropan-2-amine (0.986 ml,5.65 mmol) was added and the mixture was shaken overnight at rt on amini shaker. After 20 h the mixture was diluted with 2 ml of methanol,and shaken for 2 hr to quench any unreacted chlorotrityl resin. Theresin was vacuum filtered in a polypropylene reaction tube and washedwith 100 ml DMF, 100 ml dichloromethane, and finally 10 ml diethylether. Resin was air dried and used as is assuming a 0.44 mmol/gloading.

Preparation of Modified 2-chlorotrityl Chloride Resin B

To a 40 mL vial was added 2-chlorotrityl chloride resin (1.42 mmol/gloading) (2.129 g, 2.98 mmol). The resin was swelled in 15 mldichloromethane for 10 minutes. A solution ofS)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-undecanamidohexanoicacid (0.5 g, 0.932 mmol), in 2 ml dichloromethane followed byN-ethyl-N-isopropylpropan-2-amine (1.06 ml, 6.06 mmol) was added and themixture was shaken overnight at room temperature on a mini shaker. After20 h the mixture was diluted with 2 ml of methanol, and shaken for 2 hrto quench any unreacted chlorotrityl resin. The resin was vacuumfiltered in a polypropylene reaction tube and washed with 100 ml DMF,100 ml dichloromethane, and finally 10 ml diethyl ether. Resin was airdried and used as is assuming a 0.44 mmol/g loading.

Preparation of Example 11001

Example 11001 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetyl acid coupling procedure”, “Global DeprotectionMethod A”, and “Cyclization Method A”. Modified Rink resin D was used inthis synthesis.

The crude material was purified via preparative LC/MS with the followingconditions: Column: Waters XBridge C18, 30×100 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 30-100% B over 25 minutes, then a 5-minute hold at 100% B;Flow: 30 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 5.2mg, and its estimated purity by LCMS analysis was 98%. Analysis LCMSCondition A: Retention time=7.34 min; ESI-MS(+) m/z 1105.4 (M+2H);ESI-HRMS(+) m/z: Calculated: 1105.6498 (M+2H) Found: 1105.6494 (M+2H).

Preparation of Example 11002

Example 11002 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinD was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 3.9 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition A: Retention time=7.10 min; ESI-MS(+) m/z 1133.5(M+2H); ESI-HRMS(+) m/z: Calculated: 1133.1543 (M+2H) Found: 1133.1496(M+2H).

Preparation of Example 11003

Example 11003 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinA was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 3.5 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition A: Retention time=4.66 min; ESI-MS(+) m/z 1200.4(M+2H).

Preparation of Example 11004

Example 11004 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinA was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 10.7 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition A: Retention time=4.73 min; ESI-MS(+) m/z 1171.9(M+2H); ESI-HRMS(+) m/z: Calculated: 1171.1299 (M+2H) Found: 1171.1302(M+2H).

Preparation of Example 11005

Example 11005 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinA was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 4.8 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition A: Retention time=4.78 min; ESI-MS(+) m/z 1142.5(M+2H); ESI-HRMS(+) m/z: Calculated: 1142.6192 (M+2H) Found: 1142.6193(M+2H).

Preparation of Example 11006

Example 11006 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinA was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 9.2 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition A: Retention time=4.66 min; ESI-MS(+) m/z 1161.7(M+2H); ESI-HRMS (+) m/z: Calculated: 1161.6088 (M+2H) Found: 1161.6075(M+2H).

Preparation of Example 11007

Example 11007 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinA was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 16.0 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition A: Retention time=4.64 min; ESI-MS(+) m/z 1191.0(M+2H); ESI-HRMS(+) m/z: Calculated: 1090.0797 (M+2H) Found: 1090.0779(M+2H).

Preparation of Example 11008

Example 11008 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinA was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 12.2 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition A: Retention time=4.55 min; ESI-MS(+) m/z 1133.3(M+2H); ESI-HRMS(+) m/z: Calculated: 1133.0981 (M+2H) Found: 1133.0950(M+2H).

Preparation of Example 11009

Example 11009 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinA was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 19.8 mg, and its estimated purity by LCMSanalysis was 96%.

Analysis LCMS Condition A: Retention time=4.81 min; ESI-MS(+) m/z 1147.7(M+2H); ESI-HRMS(+) m/z: Calculated: 1147.5875 (M+2H) Found: 1147.5867(M+2H).

Preparation of Example 11010

Example 11010 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinA was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 19.4 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition A: Retention time=4.83 min; ESI-MS(+) m/z 1154.9(M+2H); ESI-HRMS(+) m/z: Calculated: 1154.5954 (M+2H) Found: 1154.9533(M+2H).

Preparation of Example 11011

Example 11011 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composes of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinA was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 8.7 mg, and its estimated purity by LCMSanalysis was 96%.

Analysis LCMS Condition A: Retention time=4.37 min; ESI-MS(+) m/z 1154.8(M+2H); ESI-HRMS(+) m/z: Calculated: 1154.5954 (M+2H) Found: 1154.5941(M+2H).

Preparation of Example 11012

Example 11012 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinA was used in this synthesis.FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid was used withthe “Custom amino acids-coupling procedure”. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 8.7 mg, and its estimatedpurity by LCMS analysis was 96%. Analysis LCMS Condition A: Retentiontime=4.68 min; ESI-MS(+) m/z 1265.3 (M+2H); ESI-HRMS(+) m/z: Calculated:1264.6791 (M+2H).

Found: 1264.6764 (M+2H).

Preparation of Example 11013

Example 11013 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinB was used in this synthesis.FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid was used withthe “Custom amino acids-coupling procedure”. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 12.7 mg, and its estimatedpurity by LCMS analysis was 98%. Analysis LCMS Condition A: Retentiontime=5.04 min; ESI-MS(+) m/z 1351.2 (M+2H).

Preparation of Example 11014

Example 11014 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinB was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 7.7 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition A:

Retention time=4.97 min; ESI-MS(+) m/z 1241.1 (M+2H).

Preparation of Example 11015

Example 11015 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinB was used in this synthesis.FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid was used withthe “Custom amino acids-coupling procedure”. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 30-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 7.1 mg, and its estimatedpurity by LCMS analysis was 99%. Analysis LCMS Condition A: Retentiontime=5.44 min; ESI-MS(+) m/z 1292.7 (M+2H); ESI-HRMS(+) m/z: Calculated:1292.7160 (M+2H).

Found: 1292.7148 (M+2H).

Preparation of Example 11016

Example 11016 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinB was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 8.3 mg, and its estimated purity by LCMSanalysis was 98%; Analysis LCMS Condition A: Retention time=5.27 min;ESI-MS(+) m/z 1182.7 (M+2H)

Preparation of Example 11017

Example 11017 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinB was used in this synthesis.FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid was used withthe “Custom amino acids-coupling procedure”. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 30-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 5.2 mg, and its estimatedpurity by LCMS analysis was 95%. Analysis LCMS Condition A: Retentiontime=4.81 min; ESI-MS(+) m/z 1408.1 (M+2H); ESI-HRMS(+) m/z: Calculated:1407.7431 (M+2H).

Found: 1407.7430 (M+2H).

Preparation of Example 11018

Example 11018 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinC was used in this synthesis.FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid was used withthe “Custom amino acids-coupling procedure”. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 30-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 11.2 mg, and its estimatedpurity by LCMS analysis was 96%. Analysis LCMS Condition A: Retentiontime=5.16 min; ESI-MS(+) m/z 1421.8 (M+2H).

Preparation of Example 11019

Example 11019 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Chloroacetic acid couplingprocedure A”, “Global Deprotection Method A”, and “Cyclization MethodA”. Modified Rink resin C was used in this synthesis. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 30-100% Bover 25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 40.2 mg, and itsestimated purity by LCMS analysis was 99%. Analysis LCMS Condition A:Retention time=6.11 min; ESI-MS(+) m/z 1091.45 (M+2H).

Preparation of Example 11020

Example 11020 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinC was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 41.0 mg, and its estimated purity by LCMSanalysis was 97%.

Analysis LCMS Condition A: Retention time=5.93 min; ESI-MS(+) m/z1119.83 (M+2H).

Preparation of Example 11021

Example 11021 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinC was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 14.4 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition A: Retention time=6.36 min; ESI-MS(+) m/z 1265.9(M+2H).

Preparation of Example 11022

Example 11022 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinC was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 5.7 mg, and its estimated purity by LCMSanalysis was 97%.

Analysis LCMS Condition A: Retention time=6.13 min; ESI-MS(+) m/z1250.51 (M+2H); ESI-HRMS(+) m/z: Calculated: 1249.1930 (M+2H) Found:1249.1934 (M+2H).

Preparation of Example 11023

Example 11023 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinC was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 3.8 mg, and its estimated purity by LCMSanalysis was 95%.

Analysis LCMS Condition A: Retention time=6.41 min; ESI-MS(+) m/z 1235.4(M+2H); ESI-HRMS(+) m/z: Calculated: 1235.6878 (M+2H) Found: 1235.6832(M+2H).

Preparation of Example 11024

Example 11024 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinC was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 7.2 mg, and its estimated purity by LCMSanalysis was 97%.

Analysis LCMS Condition A: Retention time=5.68 min; ESI-MS(+) m/z 1220.5(M+2H); ESI-HRMS(+) m/z: Calculated: 1220.6823 (M+2H) Found: 1220.6810(M+2H).

Preparation of Example 11025

Example 11025 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinA was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 3.8 mg, and its estimated purity by LCMSanalysis was 95%.

Analysis LCMS Condition A: Retention time=5.74 min; ESI-MS(+) m/z 1234.6(M+2H); ESI-HRMS(+) m/z: Calculated: 1234.6798 (M+2H) Found: 1234.6796.

Preparation of Example 11026

Example 11026 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinC was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 11.7 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition A: Retention time=5.81 min; ESI-MS(+) m/z 1206.0(M+2H); ESI-HRMS(+) m/z: Calculated: 1206.1690 (M+2H) Found: 1206.1690(M+2H).

Preparation of Example 11027

Example 11027 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinC was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 9.3 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition A: Retention time=5.92 min; ESI-MS(+) m/z 1177.6(M+2H); ESI-HRMS(+) m/z: Calculated: 1177.6583 (M+2H) Found: 1177.6585(M+2H).

Preparation of Example 11028

Example 11028 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinC was used in this synthesis.FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid was used withthe “Custom amino acids-coupling procedure”. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 7.8 mg, and its estimatedpurity by LCMS analysis was 98%. Analysis LCMS Condition A: Retentiontime=5.57 min; ESI-MS(+) m/z 1363.2 (M+2H).

Preparation of Example 11029

Example 11029 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinC was used in this synthesis.FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid was used withthe “Custom amino acids-coupling procedure”. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 16.7 mg, and its estimatedpurity by LCMS analysis was 99%. Analysis LCMS Condition A: Retentiontime=6.01 min; ESI-MS(+) m/z 1306.8 (M+2H).

Preparation of Example 11030

Example 11030 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinC was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 7.2 mg, and its estimated purity by LCMSanalysis was 95%. Analysis LCMS Condition A: Retention time=5.40 min;ESI-MS(+) m/z 1153.4 (M+2H).

Preparation of Example 11031

Example 11031 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinC was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 30-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 13.0 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition A: Retention time=5.88 min; ESI-MS(+) m/z 1125.1(M+2H).

Preparation of Example 11032

Example 11032 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Rink resin was usedin this synthesis. FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoicacid was used with the “Custom amino acids-coupling procedure”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: Waters XBridge C18, 30×100 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 15-100% B over 25 minutes, then a 5-minute hold at 100% B;Flow: 30 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 14.4mg, and its estimated purity by LCMS analysis was 98%. Analysis LCMSCondition A: Retention time=2.99 min; ESI-MS(+) m/z 1442.3 (M+2H).

Preparation of Example 11033

Example 11034 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure B”, “GlobalDeprotection Method C”, and “Cyclization Method X”. Rink resin was usedin this synthesis. FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoicacid was used with the “Custom amino acids-coupling procedure”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: Waters XBridge C18, 30×100 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 15-100% B over 25 minutes, then a 5-minute hold at 100% B;Flow: 30 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 20.7mg, and its estimated purity by LCMS analysis was 99%. Analysis LCMSCondition A: Retention time=3.26 min; ESI-MS(+) m/z 1181.4 (M+2H);ESI-HRMS(+) m/z: Calculated: 1181.0772 (M+2H)

Found: 1181.0757 (M+2H).

Preparation of Example 11034

Example 11034 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Rink resin was usedin this synthesis. FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoicacid was used with the “Custom amino acids-coupling procedure”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: Waters XBridge C18, 30×100 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 15-100% B over 25 minutes, then a 5-minute hold at 100% B;Flow: 30 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 20.8mg, and its estimated purity by LCMS analysis was 98%. Analysis LCMSCondition A: Retention time=3.63 min; ESI-MS(+) m/z 1168.0 (M+2H);ESI-HRMS(+) m/z: Calculated: 1167.0616 (M+2H).

Found: 1167.0624 (M+2H).

Preparation of Example 11035

Example 11035 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Rink resin was usedin this synthesis. FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoicacid was used with the “Custom amino acids-coupling procedure”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: Waters XBridge C18, 30×100 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 15-100% B over 25 minutes, then a 5-minute hold at 100% B;Flow: 30 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 33.2mg, and its estimated purity by LCMS analysis was 98%. Analysis LCMSCondition A: Retention time=3.63 min; ESI-MS(+) m/z 1138.4 (M+2H);ESI-HRMS(+) m/z: Calculated: 1138.5517 (M+2H).

Found: 1138.5508 (M+2H).

Preparation of Example 11036

Example 11036 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Rink resin was usedin this synthesis. FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoicacid was used with the “Custom amino acids-coupling procedure”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: Waters XBridge C18, 30×100 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 15-100% B over 25 minutes, then a 5-minute hold at 100% B;Flow: 30 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 12.2mg, and its estimated purity by LCMS analysis was 98%. Analysis LCMSCondition A: Retention time=3.88 min; ESI-MS(+) m/z 1081.9 (M+2H);ESI-HRMS(+) m/z: Calculated: 1081.0374 (M+2H) Found: 1081.0358 (M+2H).

Preparation of Example 11037

Example 11037 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Rink resin was usedin this synthesis. FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoicacid was used with the “Custom amino acids-coupling procedure”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: Waters XBridge C18, 30×100 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 15-100% B over 25 minutes, then a 5-minute hold at 100% B;Flow: 30 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 11.4mg, and its estimated purity by LCMS analysis was 98%. Analysis LCMSCondition A: Retention time=3.91 min; ESI-MS(+) m/z 1109.6 (M+2H);ESI-HRMS(+) m/z: Calculated: 1109.5481 (M+2H)

Found: 1109.5472 (M+2H).

Preparation of Example 11038

Example 11038 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Rink resin was usedin this synthesis. FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoicacid was used with the “Custom amino acids-coupling procedure”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: Waters XBridge C18, 30×100 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 15-100% B over 25 minutes, then a 5-minute hold at 100% B;Flow: 30 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 18.7mg, and its estimated purity by LCMS analysis was 98%. Analysis LCMSCondition A: Retention time=3.55 min; ESI-MS(+) m/z 1182.1 (M+2H);ESI-HRMS(+) m/z: Calculated: 1181.0828 (M+2H) Found: 1181.0816 (M+2H).

Preparation of Example 11039

Example 11039 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Rink resin was usedin this synthesis. FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoicacid was used with the “Custom amino acids-coupling procedure”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: Waters XBridge C18, 30×100 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 15-100% B over 25 minutes, then a 5-minute hold at 100% B;Flow: 30 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 13.7mg, and its estimated purity by LCMS analysis was 98%. Analysis LCMSCondition A: Retention time=3.69 min; ESI-MS(+) m/z 1123.6 (M+2H);ESI-HRMS(+) m/z: Calculated: 1123.5677 (M+2H)

Found: 1123.5694 (M+2H).

Preparation of Example 11040

Example 11040 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Rink resin was usedin this synthesis. FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoicacid was used with the “Custom amino acids-coupling procedure”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: Waters XBridge C18, 30×100 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 15-100% B over 25 minutes, then a 5-minute hold at 100% B;Flow: 30 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 19.5mg, and its estimated purity by LCMS analysis was 98%. Analysis LCMSCondition A: Retention time=3.69 min; ESI-MS(+) m/z 1096.0 (M+2H);ESI-HRMS(+) m/z: Calculated: 1095.0586 (M+2H)

Found: 1095.0567 (M+2H).

Preparation of Example 11041

Example 11041 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Rink resin was usedin this synthesis. FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoicacid was used with the “Custom amino acids-coupling procedure”. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: Waters XBridge C18, 30×100 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 15-100% B over 25 minutes, then a 5-minute hold at 100% B;Flow: 30 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 21.7mg, and its estimated purity by LCMS analysis was 97%. Analysis LCMSCondition A: Retention time=3.50 min; ESI-MS(+) m/z 1153.7 (M+2H);ESI-HRMS(+) m/z: Calculated: 1152.5721 (M+2H)

Found: 1153.5719 (M+2H).

Preparation of Example 11042

Example 11042 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin A was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 21.3 mg, and its estimatedpurity by LCMS analysis was 98%.

Analysis LCMS Condition A: Retention time=3.66 min; ESI-MS(+) m/z 1081.5(M+2H); ESI-HRMS(+) m/z: Calculated: 1081.5294 (M+2H) Found: 1081.5288(M+2H).

Preparation of Example 11043

Example 11043 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin A was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 20.7 mg, and its estimatedpurity by LCMS analysis was 97%.

Analysis LCMS Condition A: Retention time=3.66 min; ESI-MS(+) m/z 1110.8(M+2H); ESI-HRMS(+) m/z: Calculated: 1110.0401 (M+2H) Found: 1110.0392(M+2H).

Preparation of Example 11044

Example 11044 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin A was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 10.7 mg, and its estimatedpurity by LCMS analysis was 97%. Analysis LCMS Condition A: Retentiontime=3.66 min; ESI-MS(+) m/z 1117.1 (M+2H); ESI-HRMS(±) m/z: Calculated:1117.0479 (M+2H) Found: 1117.0452 (M+2H).

Preparation of Example 11045

Example 11045 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin A was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 16.0 mg, and its estimatedpurity by LCMS analysis was 98%.

Analysis LCMS Condition A: Retention time=3.66 min; ESI-MS(+) m/z 1085.1(M+2H).

Preparation of Example 11046

Example 11046 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin A was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 21.4 mg, and its estimatedpurity by LCMS analysis was 98%.

Analysis LCMS Condition A: Retention time=3.48 min; ESI-MS(+) m/z 1084.5(M+2H).

Preparation of Example 11047

Example 11047 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin A was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 20.7 mg, and its estimatedpurity by LCMS analysis was 98%.

Analysis LCMS Condition A: Retention time=3.44 min; ESI-MS(+) m/z 1084.4(M+2H); ESI-HRMS(+) m/z: Calculated: 1084.5346 (M+2H) Found: 1084.5362(M+2H).

Preparation of Example 11060

Example 11060 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin was used in this synthesis.FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid was used withthe “Custom amino acids-coupling procedure”. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 22.4 mg, and its estimatedpurity by LCMS analysis was 99%.

Analysis LCMS Condition A: Retention time=3.83 min; ESI-MS(+) m/z 1177.0(M+2H).

Preparation of Example 11061

Example 11061 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin was used in this synthesis.FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid was used withthe “Custom amino acids-coupling procedure”. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 28.3 mg, and its estimatedpurity by LCMS analysis was 99%.

Analysis LCMS Condition A: Retention time=3.97 min; ESI-MS(+) m/z 1182.5(M+2H); ESI-HRMS(+) m/z: Calculated: 1182.6316 (M+2H) Found: 1182.6275(M+2H).

Preparation of Example 11062

Example 11062 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin B was used in this synthesis.FMOC-21-Amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid was used withthe “Custom amino acids-coupling procedure”. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-100% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 2.8 mg, and its estimatedpurity by LCMS analysis was 98%. Analysis LCMS Condition A: Retentiontime=4.36 min; ESI-MS(+) m/z 1232.5 (M+2H).

Preparation of Example 11063

Example 11063 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-100% Bover 25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 27.4 mg, and itsestimated purity by LCMS analysis was 98%. Analysis LCMS Condition A:Retention time=4.11 min; ESI-MS(+) m/z 1139.7 (M+2H); ESI-HRMS(+) m/z:Calculated: 1139.6265 (M+2H) Found: 1139.6252 (M+2H).

Preparation of Example 11064

Example 11064 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% Bover 25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 26 mg, and itsestimated purity by LCMS analysis was 98.7%; Analysis LCMS Condition A:Retention time=3.90 min; ESI-MS(+) m/z 1019.9 (M+2H); ESI-HRMS(+) m/z:Calculated: 1019.0426 (M+2H) Found: 1019.0407 (M+2H).

Preparation of Example 11065

Example 11065 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% Bover 25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 37 mg, and itsestimated purity by LCMS analysis was 97.3%. Analysis LCMS Condition A:Retention time=3.706 min; ESI-MS(+) m/z 1158.0 (M+2H); ESI-HRMS(+) m/z:Calculated: 1157.0723 (M+2H) Found: 1157.0697 (M+2H).

Preparation of Example 11066

Example 11066 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% Bover 25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 48 mg, and itsestimated purity by LCMS analysis was 98.5%. Analysis LCMS Condition A:Retention time=3.578 min; ESI-MS(+) m/z 1098.7 (M+2H); ESI-HRMS(+) m/z:Calculated: 1098.0533 (M+2H) Found: 1098.0513 (M+2H).

Preparation of Example 11067

Example 11067 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% Bover 25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 36.3 mg, and itsestimated purity by LCMS analysis was 99%. Analysis LCMS Condition A:Retention time=3.225 min; ESI-MS(+) m/z 1158.6 (M+2H); ESI-HRMS(+) m/z:Calculated: 1157.5643 (M+2H) Found: 1157.5622 (M+2H).

Preparation of Modified 2-chlorotrityl chloride resin C

To a 40 mL vial was added 2-chlorotrityl chloride resin (1.2 mmol/gloading) (6.37 g, 7.65 mmol). The resin was swelled in 15 mldichloromethane for 10 minutes. A solution of (1.2 g, 2.83 mmol),FMOC-added 11-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)undecanoic acidin 5 ml dichloromethane followed by N-ethyl-N-isopropylpropan-2-amine(3.45 ml, 19.83 mmol) was added and the mixture was shaken overnight atrt on a mini shaker. After 20 h the mixture was diluted with 3 ml ofmethanol, and shaken for 2 hr to quench any unreacted chlorotritylresin. The resin was vacuum filtered in a polypropylene reaction tubeand washed with 100 ml DMF, 100 ml dichloromethane, and finally 10 mldiethyl ether. The resin was air dried and used as is assuming a 0.44mmol/g loading.

Preparation of Example 11068

Example 11068 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 32 mg, and its estimatedpurity by LCMS analysis was 99.2%. Analysis LCMS Condition A: Retentiontime=3.781 min; ESI-MS(+) m/z 1200.0 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1199.1374 (M+2H) Found: 1199.1379 (M+2H).

Preparation of Example 11069

Example 11069 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 27 mg, and its estimatedpurity by LCMS analysis was 96.9%. Analysis LCMS Condition A: Retentiontime=3.598 min; ESI-MS(+) m/z 1135.2 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1134.0695 (M+2H) Found: 1134.0691 (M+2H).

Preparation of Example 11070

Example 11070 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 31 mg, and its estimatedpurity by LCMS analysis was 98.4%. Analysis LCMS Condition A: Retentiontime=3.715 min; ESI-MS(+) m/z 1106.0 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1105.0668 (M+2H) Found: 1105.0663 (M+2H).

Preparation of Example 11071

Example 11071 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 32 mg, and its estimatedpurity by LCMS analysis was 99.2%. Analysis LCMS Condition A: Retentiontime=3.733 min; ESI-MS(+) m/z 1077.3 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1076.5561 (M+2H) Found: 1076.5547 (M+2H).

Preparation of Example 11072

Example 11072 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 51 mg, and its estimatedpurity by LCMS analysis was 98.6%. Analysis LCMS Condition A: Retentiontime=3.655 min; ESI-MS(+) m/z 1035.0 (M+2H); ESI-HRMS(+) m/z:Calculated: 1034.0297 (M+2H) Found: 1034.0269 (M+2H).

Preparation of Example 11073

Example 11073 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 29 mg, and its estimatedpurity by LCMS analysis was 98.7%. Analysis LCMS Condition A: Retentiontime=4.038 min; ESI-MS(+) m/z 1157.6 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1156.6054 (M+2H) Found: 1156.6029 (M+2H).

Preparation of Example 11074

Example 11074 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 29 mg, and its estimatedpurity by LCMS analysis was 98.7%. Analysis LCMS Condition A: Retentiontime=4.038 min; ESI-MS(+) m/z 1157.6 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1156.6054 (M+2H) Found: 1156.6029 (M+2H).

Preparation of Example 11075

Example 11075 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 40 mg, and its estimatedpurity by LCMS analysis was 98.1%. Analysis LCMS Condition A: Retentiontime=3.878 min; ESI-MS(+) m/z 1193.2 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1192.1240 (M+2H) Found: 1192.1227 (M+2H).

Preparation of Example 11076

Example 11076 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 42 mg, and its estimatedpurity by LCMS analysis was 100%. Analysis LCMS Condition A: Retentiontime=4.088 min; ESI-MS(+) m/z 1042.1 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1041.0319 (M+2H) Found: 1041.0309 (M+2H).

Preparation of Example 11077

Example 11077 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 38 mg, and its estimatedpurity by LCMS analysis was 100%. Analysis LCMS Condition A: Retentiontime=4.050 min; ESI-MS(+) m/z 1070.4 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1069.5426 (M+2H) Found: 1069.5405 (M+2H).

Preparation of Example 11078

Example 11078 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 40 mg, and its estimatedpurity by LCMS analysis was 100%. Analysis LCMS Condition A: Retentiontime=3.903 min; ESI-MS(+) m/z 1098.7 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1098.0533 (M+2H) Found: 1098.0508 (M+2H).

Preparation of Example 11079

Example 11079 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 46 mg, and its estimatedpurity by LCMS analysis was 98.9%. Analysis LCMS Condition A: Retentiontime=3.533 min; ESI-MS(+) m/z 1127.7 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1126.5641 (M+2H) Found: 1126.5608 (M+2H).

Preparation of Example 11080

Example 11080 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 27 mg, and its estimatedpurity by LCMS analysis was 99.0%. Analysis LCMS Condition A: Retentiontime=3.235 min; ESI-MS(+) m/z 1194.8 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1193.1136 (M+2H) Found: 1193.1127 (M+2H).

Preparation of Example 11081

Example 11081 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 38 mg, and its estimatedpurity by LCMS analysis was 98.5%. Analysis LCMS Condition A: Retentiontime=3.743 min; ESI-MS(+) m/z 1167.8 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1166.6185 (M+2H) Found: 1166.6167 (M+2H).

Preparation of Example 11082

Example 11082 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 28 mg, and its estimatedpurity by LCMS analysis was 99.3%. Analysis LCMS Condition A: Retentiontime=3.688 min; ESI-MS(+) m/z 1010.1 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1009.0106 (M+2H) Found: 1009.0103 (M+2H).

Preparation of Example 11083

Example 11083 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 31 mg, and its estimatedpurity by LCMS analysis was 98.8%. Analysis LCMS Condition A: Retentiontime=3.576 min; ESI-MS(+) m/z 1161.2 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1160.1027 (M+2H) Found: 1160.1039 (M+2H).

Preparation of Example 11084

Example 11084 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 27 mg, and its estimatedpurity by LCMS analysis was 98.2%.

Analysis LCMS Condition A: Retention time=3.625 min; ESI-MS(+) m/z1038.3 (M+2H); ESI-HRMS(+) m/z: Calculated: 1037.5213 (M+2H) Found:1037.5221 (M+2H).

Preparation of Example 11085

Example 11085 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 22 mg, and its estimatedpurity by LCMS analysis was 99.7%. Analysis LCMS Condition A: Retentiontime=3.951 min; ESI-MS(+) m/z 1104.7 (M+2H).

Preparation of Example 11086

Example 11086 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 32 mg, and its estimatedpurity by LCMS analysis was 95.8%. Analysis LCMS Condition A: Retentiontime=4.088 min; ESI-MS(+) m/z 1160.8 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1159.6107 (M+2H) Found: 1159.6104 (M+2H).

Preparation of Example 11087

Example 11087 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 22 mg, and its estimatedpurity by LCMS analysis was 99.5%. Analysis LCMS Condition A: Retentiontime=4.595 min; ESI-MS(+) m/z 1252.0 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1251.1918 (M+2H) Found: 1251.1919 (M+2H).

Preparation of Example 11088

Example 11088 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 22 mg, and its estimatedpurity by LCMS analysis was 94.1%. Analysis LCMS Condition A: 4 mingradient 1 min hold: Retention time=2.300 min; ESI-MS(+) m/z 1311.5(M+2H).

Preparation of Example 11089

Example 11089 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composes of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 23 mg, and its estimatedpurity by LCMS analysis was 87.6%. Analysis LCMS Condition A: Retentiontime=4.923 min; ESI-MS(+) m/z 1100.9 (M+2H).

Preparation of Example 11090

Example 11090 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 21 mg, and its estimatedpurity by LCMS analysis was 98.5%; Analysis LCMS Condition A: Retentiontime=5.395 min; ESI-MS(+) m/z 1192.7 (M+2H).

Preparation of Example 11091

Example 11091 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 27 mg, and its estimatedpurity by LCMS analysis was 98.9%. Analysis LCMS Condition A: Retentiontime=4.256 min; ESI-MS(+) m/z 1093.9 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1093.0794 (M+2H) Found: 1093.0779 (M+2H).

Preparation of Example 11092

Example 11092 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composes of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 31 mg, and its estimatedpurity by LCMS analysis was 95.9%. Analysis LCMS Condition A: Retentiontime=4.031 min; ESI-MS(+) m/z 1148.4 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1159.6107 (M+2H) Found: 1159.6104 (M+2H).

Preparation of Example 11093

Example 11093 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 16 mg, and its estimatedpurity by LCMS analysis was 95.6%. Analysis LCMS Condition A: 9 mingradient 1 min hold: Retention time=3.920 min; ESI-MS(+) m/z 1101.9(M+2H); ESI-HRMS(+) m/z: Calculated: 1101.0586 (M+2H) Found: 1101.0578(M+2H).

Preparation of Example 11094

Example 11094 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 23 mg, and its estimatedpurity by LCMS analysis was 98.3%. Analysis LCMS Condition A: 9 mingradient 1 min hold: Retention time=4.023 min; ESI-MS(+) m/z 1101.5(M+2H).

Preparation of Example 11095

Example 11095 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 25 mg, and its estimatedpurity by LCMS analysis was 98.4%. Analysis LCMS Condition A: 4 mingradient 1 min hold: Retention time=2.288 min; ESI-MS(+) m/z 1101.9(M+2H).

Preparation of Example 11096

Example 11096 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 13 mg, and its estimatedpurity by LCMS analysis was 100%. Analysis LCMS Condition A: Retentiontime=4.056 min; ESI-MS(+) m/z 1094.4 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1093.5714 (M+2H) Found: 1093.5685 (M+2H).

Preparation of Example 11097

Example 11097 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 39 mg, and its estimatedpurity by LCMS analysis was 94.4%. Analysis LCMS Condition A: Retentiontime=3.953 min; ESI-MS(+) m/z 1102.3 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1101.5506 (M+2H) Found: 1101.5499 (M+2H).

Preparation of Example 11098

Example 11098 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 17 mg, and its estimatedpurity by LCMS analysis was 98.5%. Analysis LCMS Condition A: Retentiontime=3.878 min; ESI-MS(+) m/z 1127.9 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1127.0561 (M+2H) Found: 1127.0564 (M+2H)

Preparation of Example 11099

Example 11099 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 33 mg, and its estimatedpurity by LCMS analysis was 96.3%. Analysis LCMS Condition A: Retentiontime=4.035 min; ESI-MS(+) m/z 1122.8 (M+2H).

Preparation of Example 11100

Example 11100 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 31 mg, and its estimatedpurity by LCMS analysis was 99%. Analysis LCMS Condition A: Retentiontime=4.08 min; ESI-MS(+) m/z 1122.8 (M+2H).

Preparation of Example 11101

Example 11101 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 43 mg, and its estimatedpurity by LCMS analysis was 99%. Analysis LCMS Condition A: Retentiontime=4.16 min; ESI-MS(+) m/z 1094.3 (M+2H)

Preparation of Modified 2-chlorotrityl Chloride Resin D

To a 20 ml scintillation vial was added (S)—N-FMOC-OCTYLGLYCINE (180 mg,0.440 mmol), 2-chlorotrityl chloride (1000 mg, 1.400 mmol), CH₂Cl₂ (10mL), and N-ethyl-N-isopropylpropan-2-amine (398 mg, 3.08 mmol). The vialwas sealed and shaken on a wrist action shaker overnight. The next daythe reaction was terminated by adding 2 ml methanol and shaking theflask for an additional 2 hr. The resin was then filtered and washedwith CH₂Cl₂, DMF 3×, CH₂Cl₂ 3×, and finally diethyl ether. The resin wasdried in vacuo and used as is for peptide synthesis. The resin is usedfor peptide synthesis with an assumed loading of 0.44 meq/g.

Preparation of Example 11102

Example 11102 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin D was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 30 mg, and its estimatedpurity by LCMS analysis was 98.4%. Analysis LCMS Condition A: Retentiontime=5.223 min; ESI-MS(+) m/z 1093.0 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1092.1205 (M+2H) Found: 1092.1202 (M+2H).

Preparation of Example 11103

Example 11103 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin D was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 18 mg, and its estimatedpurity by LCMS analysis was 100%. Analysis LCMS Condition A: Retentiontime=4.445 min; ESI-MS(+) m/z 1086.9 (M+2H).

Preparation of C₁₆ amine modified 4-(4-Formyl-3-methoxy-phenoxy)butyrylAM Resin A

To a 20 ml vial was added 4-(4-formyl-3-methoxy-phenoxy)butyryl AM resin(0.94 mmol/g) (2 g, 1.880 mmol), hexadecan-1-amine (1.816 g, 7.52 mmol),Sodium Triacetoxyborohydride (1.594 g, 7.52 mmol), DMF (10 mL), andAcetic Acid (0.1 mL). The vial was sealed and shaken for 48 hr on anorbital shaker. After 48 hours the reaction mixture was filtered and thecrude resin washed 5× with DMF, 3× Methanol, 5× CH₂Cl₂, and finally withDiethyl ether. The resin was dried overnight under vacuum. The loadingwas assumed to be 0.94 mmol/g and used as is in subsequent steps.

Preparation of C₁₆ amine modified 4-(4-Formyl-3-methoxy-phenoxy)butyrylAM Resin B

To a 40 ml vial was added C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin A (1 g, 0.940 mmol), 0.2M 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetic acid in DMF (9.40ml, 1.880 mmol), 0.2M HATU in DMF (9.40 ml, 1.880 mmol), and 0.2MHunig's Base in DMF (9.40 ml, 3.76 mmol). The vial was sealed andagitated on an orbital shaker overnight. The next day the reactionmixture was filtered and the crude resin washed 5× with DMF, 5× CH₂Cl₂,and finally with 2× Diethyl ether. The resin was dried overnight undervacuum. The loading was assumed to be 0.94 mmol/g and used as is insubsequent steps.

Preparation of Example 11104

Example 11104 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin B was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 5 mg, and its estimated purity by LCMS analysis was 99.5%. AnalysisLCMS Condition A: Retention time=6.025 min; ESI-MS(+) m/z 1078.25(M+2H).

Preparation of C₁₈ amine modified 4-(4-Formyl-3-methoxy-phenoxy)butyrylAM Resin A

To a 20 ml vial was added 4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin0.94 mmol/g loading (2 g, 1.880 mmol), octadecan-1-amine (2.53 g, 9.40mmol), DMF (10 mL), Acetic Acid (0.1 mL) and SodiumTriacetoxyborohydride (1.992 g, 9.40 mmol). The vial was sealed andshaken for 48 hr on an orbital shaker. After 48 hours the reactionmixture was filtered and the crude resin washed 5× with DMF, 3×Methanol, 5× CH₂Cl₂, and finally with Diethyl ether. The resin was driedovernight under vacuum. The loading was assumed to be 0.94 mmol/g andused as is in subsequent steps.

Preparation of Example 11105

Example 11105 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₈ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin A was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 5.8 mg, and its estimated purity by LCMS analysis was 99.5%.Analysis LCMS Condition A: Retention time=6.523 min; ESI-MS(+) m/z1063.85 (M+2H).

Preparation of C₁₄ amine modified 4-(4-Formyl-3-methoxy-phenoxy)butyrylAM Resin A

To a 20 ml vial was added 4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin0.94 mmol/g loading (2 g, 1.880 mmol), DMF (15 mL), tetradecan-1-amine(1.204 g, 5.64 mmol), Sodium triacetoxyborohydride (1.594 g, 7.52 mmol),and Acetic Acid (0.1 mL). The vial was sealed and shaken overnight on anorbital shaker. The next day the resin was filtered and washed 3× withmethanol, 3× with DMF, 3× with CH₂Cl₂, and finally 1× with Et₂O. Theresin was dried under vacuum and used as is in subsequent rxns. Theloading was assumed to be 0.94 mmol/g and used as is in subsequentsteps.

Preparation of C₁₄ amine modified 4-(4-Formyl-3-methoxy-phenoxy)butyrylAM Resin B

To a 40 ml vial was added C₁₄ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin A (1 g, 0.940 mmol), 0.2M 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetic acid in DMF (9.40ml, 1.880 mmol) (99026-115), 0.2M HATU (9.40 ml, 1.880 mmol) in DMF, and0.2 M Hunig's Base in DMF (9.40 ml, 3.76 mmol). The vial was sealed andshaken on an orbital shaker overnight. The next day the resin wasfiltered off and washed 3× with DMF, 3× with CH₂Cl₂, 3× with DMF, 1×with CH₂Cl₂, and finally 1× with Et₂O. The crude resin was dried underhigh vacuum. The loading was assumed to be 0.94 mmol/g and used as is insubsequent steps.

Preparation of Example 11106

Example 11106 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₄ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin B was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 13 mg, and its estimated purity by LCMS analysis was 98.9%. AnalysisLCMS Condition A: Retention time=5.688 min; ESI-MS(+) m/z 1036.00(M+2H); ESI-HRMS(+) m/z: Calculated: 1035.0921 (M+2H) Found: 1035.0925(M+2H).

Preparation of Example 11107

Example 11107 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₄ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin B was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 12 mg, and its estimated purity by LCMS analysis was 99.3%. AnalysisLCMS Condition A: Retention time=5.883 min; ESI-MS(+) m/z 1077.47(M+2H); ESI-HRMS(+) m/z: Calculated: 1076.6288 (M+2H) Found: 1076.6286(M+2H),g

Preparation of Example 11108

Example 11108 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composes of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin B was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 17 mg, and its estimated purity by LCMS analysis was 92%. AnalysisLCMS Condition A: Retention time=6.185 min; ESI-MS(+) m/z 1127.9 (M+2H);ESI-HRMS(+) m/z: Calculated: 1127.6321 (M+2H)

Found: 1127.6329 (M+2H).

Preparation of Example 11109

Example 11109 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composes of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin B was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 23 mg, and its estimated purity by LCMS analysis was 91%. AnalysisLCMS Condition A: Retention time=6.091 min; ESI-MS(+) m/z 1092.87(M+2H); ESI-HRMS(+) m/z: Calculated: 1092.1079 (M+2H)

Found: 1092.1081 (M+2H).

Preparation of Example 11110

Example 11100 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composes of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin B was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 28 mg, and its estimated purity by LCMS analysis was 94%. AnalysisLCMS Condition A: Retention time=6.488 min; ESI-MS(+) m/z 1125.34(M+2H); ESI-HRMS(+) m/z: Calculated: 1124.6268 (M+2H)

Found: 1124.6228 (M+2H).

Preparation of Example 11111

Example 11111 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composes of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin B was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 12.9 mg, and its estimated purity by LCMS analysis was 96%. AnalysisLCMS Condition A: Retention time=6.473 min; ESI-MS(+) m/z 1140.55(M+2H); ESI-HRMS(+) m/z: Calculated: 1139.6321 (M+2H)

Found: 1139.6274 (M+2H).

Preparation of C₁₆ amine modified 4-(4-Formyl-3-methoxy-phenoxy)butyrylAM Resin C

To a 20 ml vial was added C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin A (600 mg, 0.540 mmol),11-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)undecanoic acid (457 mg,1.080 mmol), DMF (5 mL), 0.2 M HATU in DMF (5.40 mL, 1.080 mmol), and0.2M Hunig's Base in DMF (5.40 mL, 2.160 mmol). The vial was sealed andshaken for 24 hr on a orbital shaker. After 24 hours the reactionmixture was filtered and the crude resin washed with methanol, 5× withDMF, 5× CH₂Cl₂, and finally with Diethyl ether. The resin was driedovernight under vacuum. The loading was assumed to be 0.94 mmol/g andused as is in subsequent steps.

Preparation of Example 11112

Example 11112 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin C was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 7 mg, and its estimated purity by LCMS analysis was 98%. AnalysisLCMS Condition A: Retention time=6.958 min; ESI-MS(+) m/z 1160.02(M+2H); ESI-HRMS(+) m/z: Calculated: 1159.1865 (M+2H)

Found: 1159.1849 (M+2H).

Preparation of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((S)-5-(tert-butoxy)-5-oxo-4-tetradecanamidopentanamido)hexanoicacid

To a 250 ml round bottom flask wasadded(S)-4-amino-5-(tert-butoxy)-5-oxopentanoic acid (3 g, 14.76 mmol),CH₂Cl₂ (100 mL), tetradecanoyl chloride (4.01 g, 16.24 mmol), andHunig's Base (5.67 mL, 32.5 mmol). The flask was kept under a blanket ofnitrogen was sealed and stirred at rt for 24 hr. After 24 hours thereaction mixture was purred into a separatory funnel and washed with satammonium chloride. The aqueous layer was extracted with a 10% methanolchloroform solution 3×. The organic fractions were combined and washedwith brine. The organic layer was separated, dried over Na₂SO₄ andevaporated in vacuo affording(S)-5-(tert-butoxy)-5-oxo-4-tetradecanamidopentanoic acid (6.1 g, 14.75mmol, 100% yield) as a thick oil. This material was used as is in thenext step. ¹H NMR (400 MHz, CHLOROFORM-d) δ 4.50-4.29 (m, 1H), 3.68(quin, J=6.7 Hz, 1H), 3.09 (q, J=7.5 Hz, 1H), 2.43-2.27 (m, 2H),2.27-2.06 (m, 2H), 1.73-1.55 (m, 2H), 1.47 (s, 9H), 1.39-1.17 (m, 20H),1.00-0.80 (m, 3H).

To a 50 ml round bottom flask was added(S)-5-(tert-butoxy)-5-oxo-4-tetradecanamidopentanoic acid (2 g, 4.84mmol), DMF (10 mL), perfluorophenyl 2,2,2-trifluoroacetate (2.71 g, 9.67mmol) and PYRIDINE (0.860 mL, 10.64 mmol). The flask was sealed with aseptum and kept under a blanket of nitrogen and stirred overnight at rt.The next day the reaction was poured into a saturated citric acidsolution and extracted with CH₂Cl₂ 3×. The organic layers were combinedand washed with brine, dried over Na₂SO₄ and evaporated in vacuo. Thecrude product (S)-1-tert-butyl 5-(perfluorophenyl)2-tetradecanamidopentanedioate (2.65 g, 4.57 mmol, 95% yield) was usedas is without purification.

To a 50 ml round bottom flask was added(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoic acid(1.653 g, 4.49 mmol), DMF (15 mL), (S)-1-tert-butyl 5-(perfluorophenyl)2-tetradecanamidopentanedioate (2.6 g, 4.49 mmol), and Hunig's Base(0.940 mL, 5.38 mmol). The flask was sealed with a septum and kept undera blanket of nitrogen. The reaction was allowed to stir for 48 hr at rt.After 48 hr the reaction was homogeneous. The reaction mixture waspoured into a saturated citric acid solution, and extracted with CH₂Cl₂3×. The organic fractions were combined, dried over Na₂SO₄ andevaporated in vacuo. The crude oil was purified by silica gelchromatography eluting with first 100% CH₂Cl₂, then 5% Methanol in 95%CH₂Cl₂. The pure fractions were combined and evaporated in vacuoaffording(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((S)-5-(tert-butoxy)-5-oxo-4-tetradecanamidopentanamido)hexanoicacid (2.35 g, 2.92 mmol, 65.1% yield) as a viscous oil. ¹H NMR (400 MHz,CHLOROFORM-d) δ 7.78 (d, J=7.5 Hz, 2H), 7.62 (br. s., 2H), 7.41 (t,J=7.4 Hz, 2H), 7.32 (t, J=7.4 Hz, 2H), 6.54 (br. s., 1H), 5.72 (m, 2H),4.39 (m, 4H), 4.27-4.14 (m, 1H), 3.28 (m, 2H), 2.26 (m, 4H), 2.15 (m,2H), 1.92 (m, 2H), 1.62 (m, 4H), 1.48 (s, 9H), 1.26 (br. s., 20H), 0.89(t, J=6.8 Hz, 3H).

Preparation of Example 11114

Example 11114 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified rink resinE was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 15-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 15 mg, and its estimated purity by LCMSanalysis was 99%. Analysis LCMS Condition A: Retention time=4.978 min;ESI-MS(+) m/z 1275.18 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1274.6803 (M+2H) Found: 1274.6791 (M+2H).

Preparation of Example 11115

Example 11115 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composes of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin C was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 4 mg, and its estimated purity by LCMS analysis was 95%. AnalysisLCMS Condition A: Retention time=6.930 min; ESI-MS(+) m/z 1131.54(M+2H); ESI-HRMS(+) m/z: Calculated: 1130.6758 (M+2H)

Found: 1130.6747 (M+2H).

Preparation of Example 11116

Example 11116 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composes of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified rink resinE was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 15-100% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 25 mg, and its estimated purity by LCMSanalysis was 95.2%. Analysis LCMS Condition A: Retention time=5.106 min;ESI-MS(+) m/z 1247.0 (M+2H) ESI-HRMS(+) m/z: Calculated: 1246.1696(M+2H) Found: 1246.1684 (M+2H).

Preparation of Example 11119

Example 11119 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin C was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 18 mg, and its estimated purity by LCMS analysis was 93%. AnalysisLCMS Condition A: Retention time=5.968 min; ESI-MS(+) m/z 1189.5 (M+2H);ESI-HRMS(+) m/z: Calculated: 1188.6813 (M+2H)

Found: 1188.6821 (M+2H).

Preparation of Example 11120

Example 11120 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin C was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 12 mg, and its estimated purity by LCMS analysis was 99%. AnalysisLCMS Condition A: Retention time=5.688 min; ESI-MS(+) m/z 1183.6 (M+2H);ESI-HRMS(+) m/z: Calculated: 1182.6323 (M+2H)

Found: 1182.6317 (M+2H).

Preparation of Example 11123

Example 11123 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinE was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 5-70% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 23 mg, and its estimated purity by LCMSanalysis was 95.3%. Analysis LCMS Condition A: Retention time=4.773 min;ESI-MS(+) m/z 1276.7 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1275.6643 (M+2H) Found: 1275.6645 (M+2H).

Preparation of Example 11124

Example 11124 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin C was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 8 mg, and its estimated purity by LCMS analysis was 99%. AnalysisLCMS Condition A: Retention time=6.405 min; ESI-MS(+) m/z 1220.3 (M+2H);ESI-HRMS(+) m/z: Calculated: 1219.1894 (M+2H)

Found: 1219.1888 (M+2H).

Preparation of Example 11125

Example 11125 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified Rink resinE was used in this synthesis. The crude material was purified viapreparative LC/MS with the following conditions: Column: Waters XBridgeC18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 5-70% B over 25 minutes, then a5-minute hold at 100% B; Flow: 30 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 17 mg, and its estimated purity by LCMSanalysis was 95%. Analysis LCMS Condition A: Retention time=4.780 min;ESI-MS(+) m/z 1341.8 (M+2H).

Preparation of Example 11126

Preparation of Example 11126A

Example 11126A was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Rink resin was usedin this synthesis. The crude material was purified via preparative LC/MSwith the following conditions: Column: Waters XBridge C18, 30×100 mm,5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10-mMammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mMammonium acetate; Gradient: 5-70% B over 25 minutes, then a 5-minutehold at 100% B; Flow: 30 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 27 mg, and its estimated purity by LCMS analysis was99%. Analysis LCMS Condition A: Retention time=3.625 min; ESI-MS(+) m/z1107.0 (M+2H).

Preparation of 1-tert-butyl 16-(perfluorophenyl) hexadecanedioate

To a 1 dram vial was added 16-(tert-butoxy)-16-oxohexadecanoic acid (100mg, 0.292 mmol), DMF (0.8 mL), perfluorophenyl 2,2,2-trifluoroacetate(164 mg, 0.584 mmol) and pyridine (0.052 mL, 0.642 mmol). The vial wassealed with a septum and stirred overnight at rt. The next day the crudereaction mixture was loaded onto a silica gel column and purified,eluting with a 5% EtOAc/95% Hexanes to 30% EtOAc/70% Hexanes. Thedesired product was the first eluting peak. very faint UV detection . .. . The pure fractions were combined and evaporated in vacuo affording1-tert-butyl 16-(perfluorophenyl) hexadecanedioate (132 mg, 0.260 mmol,89% yield) as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ 2.68 (s,2H), 2.22 (s, 2H), 1.79 (s, 2H), 1.59 (s, 4H), 1.47 (s, 9H), 1.29 (br.s., 18H).

Preparation of 11126B

To a 1 dram vial was added Example 11126A (27 mg, 0.012 mmol), DMF (0.7mL), N-ethyl-N-isopropylpropan-2-amine (9.47 mg, 0.073 mmol), and1-tert-butyl 16-(perfluorophenyl) hexadecanedioate (11.18 mg, 0.022mmol). The reaction was allowed to stir overnight at rt. The next daythe reaction was complete by LC/MS. The crude reaction was poured intodiethyl ether and a precipitate formed. This precipitate was collectedby centrifugation and the diethyl ether was decanted off. The crudesolid 14 mg was carried onto next step as is without purification.Analysis LCMS Condition A: Retention time=5.428 min; ESI-MS(+) m/z1269.3 (M+2H).

To a 1 dram vial was added Example 11126B (14 mg, 5.52 μmol), and 0.8 mLof Standard cleavage solution. The reaction was stirred at rt for 15 minand the reaction was checked by LC/MS. The reaction was complete and thecrude reaction mixture was poured into 15 mL of diethyl ether. Theresulting solid was collected after centrifugation. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-100% Bover 25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 6 mg, and itsestimated purity by LCMS analysis was 98.6%. Analysis LCMS Condition A:Retention time=4.040 min; ESI-MS(+) m/z 1241.1 (M+2H) ESI-HRMS(+) m/z:Calculated: 1240.1458 (M+2H)

Found: 1240.1445 (M+2H).

Preparation of Example 11128

Example 11128 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. C₁₆ amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin C was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 20 mg, and its estimated purity by LCMS analysis was 98.3%. AnalysisLCMS Condition A: Retention time=5.568 min; ESI-MS(+) m/z 1277.8 (M+2H).

Preparation of Modified 2-chlorotrityl chloride resin E

To a 20 ml scint vial was added(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoicacid (0.370 g, 0.9 mmol), 2-chlorotrityl resin 1.42 mmol/g loading(2.057 g, 2.88 mmol), CH₂Cl₂ (15 mL), and Hunig's Base (1.022 mL, 5.85mmol). The vial was sealed and shaken on an orbital shaker overnight atrt. The next day the reaction was diluted with 2 ml of methanol andshaken for an additional 2 hours. The resin was then filtered off,washed with DMF 3×, CH₂Cl₂ 4×, and finally Diethyl ether. The resin wasdried in vacuo and used as is for peptide synthesis with an assumedloading of 0.44 meq/g.

Preparation of Example 11129

Example 11129 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin E was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 48 mg, and its estimatedpurity by LCMS analysis was 95.6%. Analysis LCMS Condition A: Retentiontime=3.828 min; ESI-MS(+) m/z 1070.4 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1069.5426 (M+2H) Found: 1069.5392 (M+2H).

Preparation of Modified 2-chlorotrityl chloride Resin F

To a 20 ml scint vial was added(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoicacid (0.383 g, 0.9 mmol), 2-Chlorotrityl resin 1.42 mmol/g loading(2.057 g, 2.88 mmol), CH₂Cl₂ (15 mL), and Hunig's Base (1.022 mL, 5.85mmol). The vial was sealed and shaken on an orbital shaker overnight atrt. The next day the reaction was diluted with 2 ml of methanol andshaken for an additional 2 hours. The resin was then filtered off,washed with DMF 3×, CH₂Cl₂ 4×, and finally Diethyl ether. The resin wasdried in vacuo and used as is for peptide synthesis with an assumedloading of 0.44 meq/g.

Preparation of Example 11130

Example 11130 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin F was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 32 mg, and its estimatedpurity by LCMS analysis was 100%. Analysis LCMS Condition A: Retentiontime=3.803 min; ESI-MS(+) m/z 1077.6 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1076.5504 (M+2H) Found: 1076.5462 (M+2H).

Preparation of Example 11131

Example 11131 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin E was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 40 mg, and its estimatedpurity by LCMS analysis was 98.8%. Analysis LCMS Condition A: Retentiontime=3.720 min; ESI-MS(+) m/z 1067.0 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1066.0321 (M+2H) Found: 1066.0323 (M+2H).

Preparation of Example 11132

Example 11132 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin F was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 16 mg, and its estimatedpurity by LCMS analysis was 95.9%. Analysis LCMS Condition A: Retentiontime=3.725 min; ESI-MS(+) m/z 1074.0 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1073.0399 (M+2H) Found: 1073.0393 (M+2H).

Preparation of PEG amine modified 4-(4-Formyl-3-methoxy-phenoxy)butyrylAM Resin D

To a 50 ml pressure vessel was added4-(4-formyl-3-methoxy-phenoxy)butyryl AM resin 0.94 mmol/g loading (1.5g, 1.350 mmol), (9H-fluoren-9-yl)methyl(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamate hydrochloride(0.711 g, 1.485 mmol), sodium triacetoxyhydroborate (1.431 g, 6.75mmol), DMF (20 mL), and Acetic Acid (0.2 mL). The flask was sealed andshaken overnight on a wrist action shaker. The next day the resin wasfiltered off and washed with Methanol 2×, DMF 3×, and CH₂Cl₂ 4×. Theresin was dried overnight under vacuum. The loading was assumed to be0.94 mmol/g and used as is in the subsequent step.

Preparation of PEG amine modified 4-(4-Formyl-3-methoxy-phenoxy)butyrylAM Resin E

To a 7 ml vial was added PEG amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin D (0.222 g, 0.2 mmol),16-(tert-butoxy)-16-oxohexadecanoic acid (0.137 g, 0.400 mmol), HATU(2.000 mL, 0.400 mmol), N-ethyl-N-isopropylpropan-2-amine (2.000 mL,0.800 mmol) and DMF (4 mL). The vial was shaken on a wrist action shakerovernight. The next day the resin was filtered and washed with DMF 3×,and CH₂Cl₂ 4×, then Et₂O. The resin was dried overnight under vacuum.The loading was assumed to be 0.94 mmol/g and used as is in subsequentsteps.

Preparation of Example 11133

Example 11133 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. PEG amine modified4-(4-Formyl-3-methoxy-phenoxy)butyryl AM resin E was used in thissynthesis. The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters XBridge C18, 30×100 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient 15-100% B over 25 minutes, then a 5-minute hold at100% B; Flow: 30 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 3 mg, and its estimated purity by LCMS analysis was 99.6%. AnalysisLCMS Condition A: Retention time=5.353 min; ESI-MS(+) m/z 1167.1 (M+2H);ESI-HRMS(+) m/z: Calculated: 1166.1391 (M+2H) Found: 1166.1370 (M+2H).

Preparation of 1-tert-butyl 16-(perfluorophenyl) hexadecanedioate

To a 50 ml round bottom flask was added18-(tert-butoxy)-18-oxooctadecanoic acid (807 mg, 2.178 mmol),N,N-dimethylformamide (8 mL), pyridine (379 mg, 4.79 mmol), andperfluorophenyl 2,2,2-trifluoroacetate (1220 mg, 4.36 mmol). The flaskwas sealed with a septum and kept under a blanket of nitrogen andstirred overnight at rt. The next day the reaction was poured into asaturated citric acid solution and extracted with CH₂Cl₂ 3×. The organiclayers were combined and washed with brine, dried over Na₂SO₄ andevaporated in vacuo. The crude product 1-tert-butyl 18-(perfluorophenyl)octadecanedioate (1.1 g, 2.050 mmol, 94% yield) was used as is withoutpurification.

Preparation of(S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid

To a 20 ml scint vial was added 1-tert-butyl 18-(perfluorophenyl)octadecanedioate (500 mg, 0.932 mmol),(S)-4-amino-5-(tert-butoxy)-5-oxopentanoic acid (189 mg, 0.932 mmol),N,N-dimethylformamide (3 mL), and N-ethyl-N-isopropylpropan-2-amine (157mg, 1.211 mmol). The reaction was allowed to stir for 24 hr at rt. After24 hr the reaction was homogeneous. The reaction mixture was poured intoa saturated citric acid solution, and extracted with CH₂Cl₂ 3×. Theorganic fractions were combined, washed with brine, dried over Na₂SO₄and evaporated in vacuo. The crude oil(S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (518 mg, 0.932 mmol, 100% yield) was used as is withoutpurification. ¹H NMR (400 MHz, CHLOROFORM-d) δ 6.33 (m, 1H), 4.56 (m,1H), 2.45 (m, 2H), 2.24 (m, 5H), 1.95 (m, 1H), 1.61 (m, 5H), 1.49 (s,9H), 1.46 (s, 9H), 1.26 (m, 23H)

Preparation of (S)-1-tert-butyl 5-(perfluorophenyl)2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanedioate

To a 20 ml scint vial wasadded(S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (518 mg, 0.932 mmol),N,N-dimethylformamide (3 mL), pyridine (162mg, 2.050 mmol), and perfluorophenyl 2,2,2-trifluoroacetate (522 mg,1.864 mmol). The reaction was allowed to stir for 24 hr at it After 24hr the reaction mixture was poured into a saturated citric acidsolution, and extracted with CH₂Cl₂ 3×. The organic fractions werecombined, washed with brine, dried over Na₂SO₄ and evaporated in vacuo.The crude oil (S)-1-tert-butyl 5-(perfluorophenyl)2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanedioate (670 mg, 0.928mmol, 100% yield) was used as is in the next step.

Preparation of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanamido)hexanoicacid

To a 1 dram vial was added (S)-1-tert-butyl 5-(perfluorophenyl)2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanedioate (330 mg, 0.457mmol), N,N-Dimethylformamide (2 mL), N-ethyl-N-isopropylpropan-2-amine(177 mg, 1.372 mmol),and(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoicacid (168 mg, 0.457 mmol). The reaction was allowed to stir for 24 hr atrt. After 24 hr the reaction was homogeneous. The reaction mixture waspoured into a saturated citric acid solution, and extracted with CH₂Cl₂3×. The organic fractions were combined, dried over Na₂SO₄ andevaporated in vacuo. The crude oil was purified by silica gelchromatography eluting with first 100% CH₂Cl₂, then 5% Methanol in 95%CH₂Cl₂. The pure fractions were combined and evaporated in vacuoaffording(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanamido)hexanoicacid (106 mg, 0.117 mmol, 25.6% yield) as a viscous oil. ColumnPHENOMENEX-LUNA 2.0×30 mm 3 um particles; Mobile Phase A: 10:90methanol:water with 10 mM trifluoroacetic acid; Mobile Phase B: 90:10methanol:water with 10 mM trifluoroacetic acid; Temperature: 40° C.;Gradient: 0% B, 0-100% B over 5 minutes, then a 1.0-minute hold at 100%B; Flow: 0.8 mL/min; Detection: UV at 220 nm. Analysis LCMS: Retentiontime=5.740 min; ESI-MS(+) m/z 906.86 (M+H).

Preparation of Modified 2-chlorotrityl chloride Resin G

To a 20 ml scintillation vial was added 2-chlorotrityl resin 1.42 mmol/gloading (267 mg, 0.374 mmol), CH₂Cl₂ (4 mL),(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanamido)hexanoicacid (106 mg, 0.117 mmol), and N-ethyl-N-isopropylpropan-2-amine (106mg, 0.819 mmol). The vial was sealed and shaken on a wrist action shakerovernight. The next day the reaction was terminated by adding 2 mlmethanol and shaking the flask for an additional 2 hr. The resin wasthen filtered and washed with CH₂Cl₂, DMF 3×, CH₂Cl₂ 3× and finallydiethyl ether. The resin was dried in vacuo and used as is for peptidesynthesis with an assumed loading of 0.44 meq/g.

Preparation of Example 11134

Example 11134 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin G was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters XBridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 5-70% B over25 minutes, then a 5-minute hold at 100% B; Flow: 30 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 26 mg, and its estimatedpurity by LCMS analysis was 100%. Analysis LCMS Condition A: Retentiontime=4.280 min; ESI-MS(+) m/z 1222.0 (M+2H).

Preparation of Example 11135

Example 11135 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin D was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 24 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition A: Retention time=4.141 min.

Preparation of Example 11136

Example 11136 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 22 mg, and its estimated purity by LCMSanalysis was 95.2%. Analysis LCMS Condition A: Retention time=3.930 min;ESI-MS(+) m/z 1141.0969 (M+2H).

Preparation of Modified 2-chlorotrityl chloride resin H

Step 1

Diphenyl phosphorazidate (1.738 ml, 8.04 mmol) was added to a solutionof 18-(tert-butoxy)-18-oxooctadecanoic acid (1.49 g, 4.02 mmol) andtriethylamine (1.115 ml, 8.04 mmol) in Toluene (16.08 ml) at roomtemperature. The resulting mixture was heated under reflux overnight.The reaction mixture was cooled to room temperature and quenched with a5% citric acid solution. The mixture was concentrated to half volume invacuo and then extracted 3 times with dichloromethane. The combinedorganics were washed with brine, dried over MgSO₄, filtered, andconcentrated to give crude product. The crude product was dissolved indichloromethane and applied to a 40 g ISCO silica gel cartridge. Theproduct was eluted by a 0-25% ethyl acetate/hexanes gradient. Likefractions were combined and concentrated to give tert-butyl17-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)heptadecanoate (0.384 g,0.681 mmol, 16.94% yield) as a white solid. ¹H NMR (400 MHz,CHLOROFORM-d) δ 7.78 (d, J=7.3 Hz, 2H), 7.61 (d, J=7.5 Hz, 2H), 7.41 (t,J=7.4 Hz, 2H), 7.33 (td, J=7.4, 1.0 Hz, 2H), 4.41 (d, J=7.0 Hz, 2H),4.24 (d, J=6.8 Hz, 1H), 3.20 (q, J=6.8 Hz, 2H), 2.21 (t, J=7.5 Hz, 2H),1.61-1.48 (m, 4H), 1.45 (s, 9H), 1.26 (s, 24H).

Step 2

Trifluoroacetic acid (1.5 mL, 19.47 mmol) was added to a solution oftert-butyl 17-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)heptadecanoate(0.375 g, 0.665 mmol) in Dichloromethane (5 mL). The reaction mixtureturns dark from yellow after 2 minutes. The solution was stirred at roomtemperature for 3 hours. The reaction mixture was checked by ¹HNMR anddetermined to be complete at this time. The reaction mixture wasconcentrated in vacuo. The resulting residue was taken up in 5 mldichloromethane and concentrated again. This process was repeated 3times and the resulting yellow solid was dried under vacuum overnight.17-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)heptadecanoic acid wasisolated in quantitative yield as a yellow solid. ¹H NMR (400 MHz,CHLOROFORM-d) δ 7.78 (d, J=7.5 Hz, 2H), 7.63-7.58 (m, 2H), 7.41 (t,J=7.4 Hz, 2H), 7.33 (td, J=7.5, 1.1 Hz, 2H), 4.41 (d, J=6.8 Hz, 2H),4.27-4.19 (m, 1H), 3.25-3.09 (m, 2H), 2.36 (t, J=7.5 Hz, 2H), 1.70-1.59(m, 2H), 1.55-1.40 (m, 2H), 1.27 (br. s., 24H).

Step 3

To a 20 mL vial was added 2-Chlorotrityl chloride resin (1.605 g, 1.926mmol) and 7 ml dichloromethane to swell the resin.9-((((16-carboxyhexadecyl)carbamoyl)oxy)methyl)-9H-fluoren-1-ylium(0.305 g, 0.602 mmol) in 5 ml dichloromethane andN-ethyl-N-isopropylpropan-2-amine (0.841 ml, 4.82 mmol) were added tothe vial containing the resin. The vessel was sealed and shakenovernight at room temperature on a wrist action shaker. The next day thereaction was diluted with 5 ml of methanol, and the vessel was shakenfor 2 hr to quench any unreacted chlorotrityl resin. The resin wasfiltered, and washed with DMF three times, CH₂Cl₂ 3 times, and finallyEt₂O. The resulting resin was air dried and used as is assuming a 0.44meq/g loading.

Preparation of Modified 2-chlorotrityl chloride resin I

Modified 2-chlorotrityl chloride resin I was made following an identicalprocedure to Modified 2-chlorotrityl resin A.

Preparation of Modified 2-chlorotrityl chloride resin J

Modified 2-chlorotrityl chloride resin J was made following an identicalprocedure to Modified 2-chlorotrityl resin A.

Preparation of Modified 2-chlorotrityl chloride resin K

Modified 2-chlorotrityl chloride resin K was made following an identicalprocedure to Modified 2-chlorotrityl resin A.

Preparation of Modified 2-chlorotrityl chloride resin L

Modified 2-chlorotrityl chloride resin L was made following an identicalprocedure to Modified 2-chlorotrityl resin A.

Preparation of Modified 2-chlorotrityl chloride resin M

Step 1: Preparation of 18-(tert-butoxy)-18-oxooctadecanoic acid

A suspension of octadecanedioic acid (15 g, 47.7 mmol) in Toluene (191ml) was brought to reflux in 3 neck 1 L round bottom flask. When all ofthe acid was in solution, 1,1-di-tert-butoxy-N,N-dimethylmethanamine(22.87 ml, 95 mmol) was added dropwise over 30 minutes. The reactionmixture was heated under reflux overnight. The reaction was stoppedafter 20 total hours of heating. The reaction mixture was cooled to roomtemperature resulting in precipitation of solids. The mixture wasfiltered by vacuum filtration. The resulting white solid was suspendedin 200 ml dichloromethane and stirred for 15 minutes. The remainingsolids were removed via vacuum filtration. The collected solids wereagain suspended in dichloromethane and stirred for 15 minutes. After asecond filtration the combined filtrates were concentrated in vacuo andthe resulting white powder was dried under vacuum.18-(tert-butoxy)-18-oxooctadecanoic acid (10.14 g, 27.4 mmol, 57.4%yield) was isolated as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ2.35 (t, J=7.5 Hz, 2H), 2.21 (t, J=7.5 Hz, 2H), 1.70-1.52 (m, 4H), 1.45(s, 9H), 1.36-1.22 (m, 24H).

Step 2

To a 250 ml round bottom flask was added18-(tert-butoxy)-18-oxooctadecanoic acid (8.58 g, 23.15 mmol), pyridine(4.68 ml, 57.9 mmol), DMF (50 ml), and perfluorophenyl2,2,2-trifluoroacetate (7.96 ml, 46.3 mmol). The flask was sealed with aseptum and kept under a nitrogen atmosphere and stirred overnight atroom temperature. The reaction mixture was poured into a saturatedcitric acid solution and extracted with CH₂Cl₂ three times. The organiclayers were combined and washed with brine, dried over MgSO₄, andevaporated in vacuo. The crude product was purified on silica gelchromatography eluting with 0% ethyl acetate/100% hexanes to 55% ethylacetate/45% hexanes. The pure fractions were combined and evaporated invacuo affording 1-tert-butyl 18-(perfluorophenyl) octadecanedioate (8.37g, 15.60 mmol, 67.4% yield) as a white solid. ¹H NMR (400 MHz,CHLOROFORM-d) δ 2.66 (t, J=7.5 Hz, 2H), 2.23-2.19 (m, 2H), 1.84-1.73 (m,2H), 1.59 (d, J=7.3 Hz, 2H), 1.45 (s, 9H), 1.44-1.38 (m, 2H), 1.33-1.25(m, 24H).

Step 3

To a 500 ml round bottom flask was added 1-tert-butyl18-(perfluorophenyl) octadecanedioate (14.2 g, 26.5 mmol), H-GLU-OBZL(5.71 g, 24.06 mmol), DMF (160 ml), andN-ethyl-N-isopropylpropan-2-amine (12.60 ml, 72.2 mmol). The flask wassealed with a septum and kept under a nitrogen atmosphere and stirredovernight at room temperature. The reaction mixture was heterogeneous.After 25 hours the reaction mixture was poured into a saturated citricacid solution and extracted with CH₂Cl₂ 3×. The organic layers werecombined and washed with brine, dried over MgSO₄ and evaporated invacuo. The crude product was purified on silica gel chromatographyeluting with a 0-7% CH₂Cl₂/MeOH gradient. The pure fractions werecombined and evaporated in vacuo affording(S)-5-(benzyloxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (7.74 g, 13.12 mmol, 54.6% yield) as a white solid. ¹H NMR (400MHz, CHLOROFORM-d) δ 7.41-7.30 (m, 5H), 6.28 (d, J=7.8 Hz, 1H), 5.18 (s,2H), 4.70 (td, J=8.0, 5.0 Hz, 1H), 2.46-2.32 (m, 2H), 2.22 (q, J=7.9 Hz,5H), 2.05-1.91 (m, 1H), 1.60 (dt, J=15.3, 7.4 Hz, 4H), 1.45 (s, 9H),1.30-1.25 (m, 24H).

Step 4

To a 100 ml round bottom flask wasadded(S)-5-(benzyloxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (1.69 g, 2.87 mmol), Dichloromethane (14.33 ml),(9H-fluoren-9-yl)methyl(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamate hydrochloride(1.373 g, 2.87 mmol),2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphate(V) (1.416 g, 3.72 mmol), andN-ethyl-N-isopropylpropan-2-amine (1.497 ml, 8.60 mmol). The flask wassealed with a septum and kept under a blanket of nitrogen and stirredovernight at room temperature. The reaction was complete by LC/MS. Thesolvent was evaporated in vacuo. The crude product was purified onsilica gel chromatography eluting with 20% acetone/hexanes to 60%acetone/40% hexanes. The pure fractions were combined and evaporated invacuo affording(S)-tert-butyl22-((benzyloxy)carbonyl)-1-(9H-fluoren-9-yl)-3,19,24-trioxo-2,8,11,14-tetraoxa-4,18,23-triazahentetracontan-41-oate(2.54 g, 2.504 mmol, 87% yield) as a yellow solid. ¹H NMR (400 MHz,CHLOROFORM-d) δ 7.76 (d, J=7.5 Hz, 2H), 7.61 (d, J=7.5 Hz, 2H),7.43-7.37 (m, 2H), 7.36-7.28 (m, 7H), 6.83 (d, J=7.3 Hz, 1H), 6.54 (br.s., 1H), 5.51-5.43 (m, 1H), 5.21-5.10 (m, 2H), 4.58-4.50 (m, 1H), 4.40(d, J=7.0 Hz, 2H), 4.25-4.17 (m, 1H), 3.65-3.48 (m, 12H), 3.37-3.25 (m,4H), 2.23-2.18 (m, 7H), 2.07-1.94 (m, 1H), 1.76-1.71 (m, 2H), 1.66-1.53(m, 6H), 1.45 (s, 9H), 1.30-1.23 (m, 24H).

Step 5

To a 100 ml round bottom flask was added(S)-tert-butyl22-((benzyloxy)carbonyl)-1-(9H-fluoren-9-yl)-3,19,24-trioxo-2,8,11,14-tetraoxa-4,18,23-triazahentetracontan-41-oate(0.95 g, 0.937 mmol), Methanol (20 ml), and 10% palladium on carbon(0.100 g, 0.094 mmol). The flask was sealed with a septum and chargedwith hydrogen via a balloon. The mixture was allowed to stir overnight.The reaction was checked by LC/MS and was complete. The reaction wasfiltered through celite to remove the catalyst and the filtrate wasevaporated in vacuo to give(S)-22-(18-(tert-butoxy)-18-oxooctadecanamido)-1-(9H-fluoren-9-yl)-3,19-dioxo-2,8,11,14-tetraoxa-4,18-diazatricosan-23-oicacid (0.76 g, 0.822 mmol, 88% yield). This material was used as iswithout purification. LC/MS: (M+H)⁺=925.10.

Step 6: Modified 2-chlorotrityl Resin M

To a 75 ml peptide vessel was added 2-Chlorotrityl chloride resin (1.580g, 2.53 mmol) and Dichloromethane (15.80 ml). After 10 minutes,(S)-22-(18-(tert-butoxy)-18-oxooctadecanamido)-1-(9H-fluoren-9-yl)-3,19-dioxo-2,8,11,14-tetraoxa-4,18-diazatricosan-23-oicacid (0.73 g, 0.790 mmol), 1-chloro-4-methylbenzene (0.093 ml, 0.790mmol), and N-ethyl-N-isopropylpropan-2-amine (1.101 ml, 6.32 mmol) wereadded. The vessel was sealed and shaken on a wrist action shaker for 45minutes. LC/MS analysis of the comparison of the ratio of the internalstandard, 1-chloro-4-methylbenzene (68.1 mg, 0.538 mmol) vs startingacid indicates reaction completion or total consumption of the acid. Theresin was then diluted with 20 ml of a 9:1 Methanol/Hunigs base solutionand quickly filtered and washed with DMF three times, CH₂Cl₂ 3 times,and finally diethyl ether. The resin was dried in vacuo and used as isfor peptide synthesis with an assumed loading of 0.5 meq/g.

Preparation of Modified 2-chlorotrityl chloride resin N

Modified 2-chlorotrityl chloride resin N was made following an identicalprocedure to Modified 2-chlorotrityl resin M.

Preparation of Modified 2-chlorotrityl chloride resin O

Modified 2-chlorotrityl chloride resin O was made following an identicalprocedure to Modified 2-chlorotrityl resin M.

Preparation of Modified 2-chlorotrityl chloride resin P

Modified 2-chlorotrityl chloride resin P was made following an identicalprocedure to Modified 2-chlorotrityl resin M.

Preparation of Modified 2-chlorotrityl chloride resin Q

Modified 2-chlorotrityl chloride resin Q was made following an identicalprocedure to Modified 2-chlorotrityl resin M.

Preparation of Modified 2-chlorotrityl chloride resin R

To a peptide vessel was added5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pentanoic acid (0.118 g,0.348 mmol), Chlorotrityl resin (0.795 g, 1.113 mmol),N-ethyl-N-isopropylpropan-2-amine (0.424 mL, 2.434 mmol), and CH₂Cl₂ (6mL). The vessel was sealed and shaken on a wrist action shakerovernight. The next day the reaction was terminated by adding 3 mlmethanol and shaking the flask for an additional 2 hr. The resin wasthen filtered and washed with CH₂Cl₂, DMF 3×, CH₂Cl₂ 3× and finallydiethyl ether. The resin was dried in vacuo and used as is, the assumedloading of 0.44 meq/g was used for preparation of the desired peptides.

Preparation of Example 11137

Example 11137 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin R was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 16 mg, and its estimated purity by LCMSanalysis was 92.2%. Analysis LCMS Condition A: Retention time=4.120 min.

Preparation of Example 11138

Example 11138 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 7 mg, and its estimated purity by LCMSanalysis was 95.1%. Analysis LCMS Condition A: Retention time=4.641 min;ESI-MS(+) m/z 1159.1391 (M+2H).

Preparation of Example 11139

Example 11139 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 6 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition A: Retention time=4.286 min;ESI-MS(+) m/z 1213.6656 (M+2H).

Preparation of Example 11140

Example 11140 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin G was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 6 mg, and its estimated purity by LCMSanalysis was 97.3%. Analysis LCMS Condition A: Retention time=4.435 min.

Preparation of Modified 2-chlorotrityl chloride resin S

Step 1

To a 50 ml round bottom flask was added H-LYS(FMOC)-OH (367 mg, 0.996mmol), N,N-Dimethylformamide (8 mL), 1-tert-butyl 18-(perfluorophenyl)octadecanedioate (641 mg, 1.195 mmol), and Hunig's Base (0.522 mL, 2.99mmol). The flask was sealed with a septum and kept under a blanket ofnitrogen and stirred overnight at rt. The next day the reaction waspoured into a saturated citric acid solution and extracted with CH₂Cl₂3×. The organic layers were combined and washed with brine, dried overNa₂SO₄ and evaporated in vacuo. The crude product was purified on silicagel chromatography eluting with 100% CH₂Cl₂ then 5% MeOH in 95% CH₂Cl₂.The pure fractions were combined and evaporated in vacuo affording(S)-6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)hexanoicacid (332 mg, 0.460 mmol, 46.2% yield). ¹H NMR (400 MHz, CHLOROFORM-d) δ7.79 (d, J=7.5 Hz, 2H), 7.60 (d, J=7.3 Hz, 2H), 7.47-7.38 (m, 2H),7.37-7.30 (m, 2H), 6.44 (m, 1H), 5.00 (t, J=6.3 Hz, 1H), 4.60-4.50 (m,1H), 4.50-4.33 (m, 2H), 4.30-4.14 (m, 1H), 3.22 (m, 2H), 2.42-2.33 (m,1H), 2.22 (t, J=7.5 Hz, 4H), 1.94 (br. s., 1H), 1.80 (m, 1H), 1.71-1.51(m, 6H), 1.48-1.45 (m, 9H), 1.38-1.12 (m, 24H).

Step 2

To a peptide vessel was added Chlorotrityl resin (921 mg, 1.474 mmol),(S)-6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)hexanoicacid (332 mg, 0.460 mmol), CH₂Cl₂ (10 mL), 1-chloro-4-methylbenzene(17.49 mg, 0.138 mmol), and N-ethyl-N-isopropylpropan-2-amine (0.561 mL,3.22 mmol). The vessel was sealed and shaken on a wrist action shakerfor 30 min. The reaction was complete by analyzing the LC/MS andcomparing the ratio of the internal standard 1-chloro-4-methylbenzene(17.49 mg, 0.138 mmol) vs. starting acid. The resin was then dilutedwith 20 ml of a 9:1 Methanol/Hunigs base solution and quickly filteredand washed with DMF 3×, CH₂Cl₂ 2× and finally diethyl ether. The resinwas dried in vacuo and was used as is with an assumed loading of 0.5meq/g for the synthesis of the desired proteins.

Preparation of Example 11141

Example 11141 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin S was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 12 mg, and its estimated purity by LCMSanalysis was 95.9%. Analysis LCMS Condition A: Retention time=5.230 min;ESI-MS(+) m/z 1157.1375 (M+2H).

Preparation of Example 11142

Example 11142 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin S was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 9 mg, and its estimated purity by LCMSanalysis was 97.6%. Analysis LCMS Condition A: Retention time=4.743 min;ESI-MS(+) m/z 1279.7212 (M+2H).

Preparation of Example 11143

Example 11143 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin S was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 3 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition A: Retention time=4.895 min;ESI-MS(+) m/z 1128.6300 (M+2H).

Preparation of Example 11144

Example 11139 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 6 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition A: Retention time=4.286 min;ESI-MS(+) m/z 1213.6656 (M+2H).

Preparation of Example 11145

Example 11145 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 13 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition A: Retention time=3.505 min;ESI-MS(+) m/z 1167.6096 (M+2H).

Preparation of Example 11146

Example 11146 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin S was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 6 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition A: Retention time=4.691 min;ESI-MS(+) m/z 1214.1614 (M+2H).

Preparation of Example 11147

Example 11147 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 24 mg, and its estimated purity by LCMSanalysis was 89.3%. Analysis LCMS Condition A: Retention time=3.501 min;ESI-MS(+) m/z 1209.0971 (M+2H).

Preparation of Example 11148

Example 11148 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 14 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition A: Retention time=3.696 min;ESI-MS(+) m/z 1151.5832 (M+2H).

Preparation of Example 11149

Example 11149 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 18 mg, and its estimated purity by LCMSanalysis was 92.1%. Analysis LCMS Condition A: Retention time=3.865 min;ESI-MS(+) m/z 1051.5311 (M+2H).

Preparation of Example 11150

Example 11150 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin G was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 6 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition A: Retention time=4.425 min;ESI-MS(+) m/z 1278.6850 (M+2H).

Preparation of Example 11151

Example 11151 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 11 mg, and its estimated purity by LCMSanalysis was 98.8%. Analysis LCMS Condition A: Retention time=4.235 min;ESI-MS(+) m/z 1167.1178 (M+2H).

Preparation of Example 11152

Example 11152 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin M was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 7 mg, and its estimated purity by LCMSanalysis was 96.9%. Analysis LCMS Condition A: Retention time=4.945 min;ESI-MS(+) m/z 1230.1853 (M+2H).

Preparation of Modified 2-chlorotrityl chloride resin T

Step 1

To a 50 ml round bottom flask wasadded(S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-aminobutanoicacid (200 mg, 0.588 mmol), N,N-dimethylformamide (5 mL), 1-tert-butyl18-(perfluorophenyl) octadecanedioate (347 mg, 0.646 mmol), and Hunig'sBase (0.308 mL, 1.763 mmol). The flask was sealed with a septum and keptunder a blanket of nitrogen and stirred overnight at rt. The next daythe reaction was poured into a saturated citric acid solution andextracted with CH₂Cl₂ 3×. The organic layers were combined and washedwith brine, dried over Na₂SO₄ and evaporated in vacuo. The crude productwas purified on silica gel chromatography eluting with 100% CH₂Cl₂ then5% MeOH in 95% CH₂Cl₂. The pure fractions were combined and evaporatedin vacuo affording(S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)butanoicacid (67 mg, 0.097 mmol, 16.46% yield). Column: X-Bridge C18, 2.0×50 mm,3.5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mMammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mMammonium acetate; Temperature: 40° C.; Gradient: 0% B, 0-100% B over 5minutes, then a 1.0-minute hold at 100% B; Flow: 0.8 mL/min; Detection:UV at 220 nm. Retention time=3.866 min; ESI-MS(−) m/z 691.6 (M−H).

Step 2

To a 20 ml scintillation vial was added(S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)butanoicacid (64 mg, 0.092 mmol), Chlorotrityl resin (196 mg, 0.314 mmol),CH₂Cl₂ (4 mL), and N-ethyl-N-isopropylpropan-2-amine (0.113 mL, 0.647mmol). The vial was sealed and shaken on a wrist action shakerovernight. The next day the reaction was terminated by adding 3 mlmethanol and shaking the flask for an additional 1 hr. The resin wasthen filtered and washed with CH₂Cl₂, DMF 3×, CH₂Cl₂ 3× and finallydiethyl ether. The resin was used as is with an assumed loading of 0.5meq/g for the subsequent synthetic steps.

Step 3

To a peptide vessel was added the indicated resin (0.209 g, 0.092 mmol),DMF (3 mL), PIPERIDINE (0.182 mL, 1.840 mmol) and the vessel was sealedand shaken on a wrist action shaker for 1 h. After 1 hour, the resin wasthen filtered and washed with CH₂Cl₂, DMF 3×, CH₂Cl₂ 3× and finallydiethyl ether. The resin was dried in vacuo and used as is in the nextstep. The resin was used as is with an assumed loading of 0.5 meq/g forthe subsequent synthetic steps.

Step 4

To a 50 ml round bottom flask was added1-(9-FLUORENYLMETHYLOXYCARBONYL-AMINO)-4,7,10-TRIOXA-13-TRIDECANAMINEHYDROCHLORIDE (1 g, 2.088 mmol), THF (15 mL), Hunig's Base (0.474 mL,2.71 mmol), and 1,1′-CARBONYLDIIMIDAZOLE (0.372 g, 2.296 mmol). Thesolution was stirred under a blanket of nitrogen overnight. The next daythe reaction was checked by LC/MS and the reaction was complete. Thereaction solvent was evaporated in vacuo and the crude oil was purifiedby silica gel chromatography eluting with 3%/97% meoH/CH2CL2. The purefractions were combined and evaporated in vacuo affording(9H-fluoren-9-yl)methyl(1-(1H-imidazol-1-yl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamate(1.022 g, 1.905 mmol, 91% yield). ¹H NMR (400 MHz, CHLOROFORM-d) δ 8.18(s, 1H), 7.78 (d, J=7.5 Hz, 2H), 7.61 (d, J=7.5 Hz, 2H), 7.47 (s, 1H),7.41 (t, J=7.4 Hz, 2H), 7.32 (td, J=7.5, 0.9 Hz, 2H), 7.21 (br. s., 1H),7.05 (dd, J=1.5, 0.8 Hz, 1H), 5.57 (br. s., 1H), 4.43 (d, J=7.5 Hz, 2H),4.30-4.16 (m, 1H), 3.72-3.60 (m, 8H), 3.60-3.53 (m, 4H), 3.49 (t, J=5.3Hz, 2H), 3.30 (q, J=5.9 Hz, 2H), 1.96-1.85 (m, 2H), 1.80-1.69 (m, 2H).Column: X-Bridge C18, 2.0×50 mm, 3.5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10 mM ammonium acetate; Temperature: 40° C.;Gradient: 0% B, 0-100% B over 4 minutes, then a 1.0-minute hold at 100%B; Flow: 0.8 mL/min; Detection: UV at 220 nm. Retention time=2.538 min;ESI-MS(+) m/z 537.3 (M+H).

Step 5

To a 25 ml round bottom flask was added (9H-fluoren-9-yl)methyl(1-(1H-imidazol-1-yl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamate(400 mg, 0.745 mmol), Acetonitrile (3 mL) and IODOMETHANE (0.093 mL,1.491 mmol). The reaction was stirred under a blanket of nitrogenovernight. The next day the reaction was checked by LC/MS and wascomplete. The reaction solvent was evaporated in vacuo and the crudesolid was used as is without purification. Column: X-Bridge C18, 2.0×50mm, 3.5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mMammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mMammonium acetate; Temperature: 40° C.; Gradient: 0% B, 0-100% B over 4minutes, then a 1.0-minute hold at 100% B; Flow: 0.8 mL/min; Detection:UV at 220 nm. Retention time=3.256 min.

Step 6

To a peptide vessel was added the above modified chlorotrityl resin(0.069 g, 0.092 mmol), CH₂Cl₂ (2 mL), Hunig's Base (0.064 mL, 0.368mmol) and the Iodomethylimidazolium reagent (0.076 g, 0.138 mmol). Thevessel was sealed and shaken on a wrist action shaker overnight. Thenext day the resin was filtered and washed with CH₂Cl₂, DMF 3×, CH₂Cl₂3× and finally diethyl ether. The resin was dried in vacuo and used asis for peptide synthesis. Assumed loading of 0.44 meq/g.

Preparation of Example 11153

Example 11153 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin T was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 3.6 mg, and its estimated purity by LCMSanalysis was 97%. Analysis LCMS Condition A: Retention time=4.848 min;ESI-MS(+) m/z 1237.6914 (M+2H).

Preparation of Example 11154

Example 11154 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin C was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 15 mg, and its estimated purity by LCMSanalysis was 97.7%. Analysis LCMS Condition A: Retention time=3.581 min.

Preparation of Example 11155

Example 11155 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin M was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 3 mg, and its estimated purity by LCMSanalysis was 97.3%. Analysis LCMS Condition A: Retention time=4.100 min;ESI-MS(+) m/z 1277.7061 (M+2H).

Preparation of Example 11156

Example 11156 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin M was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 16 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition A: Retention time=4.335 min;ESI-MS(+) m/z 1258.6989 (M+2H).

Preparation of Modified 2-chlorotrityl chloride resin U

Step 1

To a 50 ml round bottom flask was added(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-aminopropanoic acid,HCl (218 mg, 0.6 mmol), N,N-dimethylformamide (6 mL), 1-tert-butyl16-(perfluorophenyl) hexadecanedioate (397 mg, 0.780 mmol), and Hunig'sBase (0.314 mL, 1.800 mmol). The flask was sealed with a septum and keptunder a blanket of nitrogen and stirred overnight at rt. The next daythe reaction was poured into a saturated citric acid solution andextracted with CH₂Cl₂ 3×. The organic layers were combined and washedwith brine, dried over Na₂SO₄ and evaporated in vacuo. The crude productwas purified on silica gel chromatography eluting with 100% CH₂Cl₂ then5% MeOH in 95% CH₂Cl₂. The pure fractions were combined and evaporatedin vacuo affording(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(16-(tert-butoxy)-16-oxohexadecanamido)propanoicacid (386 mg, 0.593 mmol, 99% yield). Column: X-Bridge C18, 2.0×50 mm,3.5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mMammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mMammonium acetate; Temperature: 40° C.; Gradient: 0% B, 0-100% B over 6minutes, then a 1.0-minute hold at 100% B; Flow: 0.8 mL/min; Detection:UV at 220 nm. Retention time=4.570 min; ESI-MS(−) m/z 649.7 (M−H).

Step 2

To a peptide vessel was added Chlorotrityl resin (1076 mg, 1.721 mmol),(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(16-(tert-butoxy)-16-oxohexadecanamido)propanoicacid (350 mg, 0.538 mmol), CH₂Cl₂ (8 mL), 1-chloro-4-methylbenzene (68.1mg, 0.538 mmol), and N-ethyl-N-isopropylpropan-2-amine (0.656 mL, 3.76mmol). The vessel was sealed and shaken on a wrist action shaker for 20min. The reaction was complete by analyzing the LC/MS and comparing theratio of the internal standard 1-chloro-4-methylbenzene (68.1 mg, 0.538mmol) vs. starting acid. The resin was then diluted with 20 ml of a 9:1

Methanol/Hunigs base solution and quickly filtered and washed with DMF3×, CH₂Cl₂ 2× and finally diethyl ether. The resin was dried in vacuoand used as is with an assumed loading of 0.5 meq/g.

Preparation of Example 11157

Example 11157 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin U was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 22 mg, and its estimated purity by LCMSanalysis was 99%. Analysis LCMS Condition A: Retention time=4.103 min;ESI-MS(+) m/z 1261.1862 (M+2H).

Preparation of Modified 2-chlorotrityl chloride resin V

Step 1

To a 100 ml round bottom flask was added16-(tert-butoxy)-16-oxohexadecanoic acid (5900 mg, 17.23 mmol),N,N-dimethylformamide (30 mL), pyridine (3.48 mL, 43.1 mmol), andperfluorophenyl 2,2,2-trifluoroacetate (9649 mg, 34.5 mmol). The flaskwas sealed with a septum and kept under a blanket of nitrogen andstirred overnight at rt. The next day the reaction was poured into asaturated citric acid solution and extracted with CH₂Cl₂ 3×. The organiclayers were combined and washed with brine, dried over Na₂SO₄ andevaporated in vacuo. The crude product 1-tert-butyl 16-(perfluorophenyl)hexadecanedioate (8.7 g, 17.11 mmol, 99% yield) was used as is withoutpurification. ¹H NMR (400 MHz, CHLOROFORM-d) δ 2.68 (t, J=7.4 Hz, 2H),2.23 (t, J=7.5 Hz, 2H), 1.89-1.71 (m, 2H), 1.65-1.54 (m, 2H), 1.47 (s,9H), 1.28 (m, 20H).

Step 2

To a 50 ml round bottom flask was added(S)-4-amino-5-(benzyloxy)-5-oxopentanoic acid (800 mg, 3.37 mmol),N,N-Dimethylformamide (8 mL), 1-tert-butyl 16-(perfluorophenyl)hexadecanedioate (2229 mg, 4.38 mmol), and Hunig's Base (1.767 mL, 10.12mmol). The flask was sealed with a septum and kept under a blanket ofnitrogen and stirred overnight at rt. The next day the reaction waspoured into a saturated citric acid solution and extracted with CH₂Cl₂3×. The organic layers were combined and washed with brine, dried overNa₂SO₄ and evaporated in vacuo. The crude product was purified on silicagel chromatography eluting with 100% CH₂Cl₂ then 5% MeOH in 95% CH₂Cl₂.The pure fractions were combined and evaporated in vacuo affording(S)-5-(benzyloxy)-4-(16-(tert-butoxy)-16-oxohexadecanamido)-5-oxopentanoicacid (0.836 g, 1.488 mmol, 44.1% yield). Analysis LCMS Condition A:Retention time=4.641 min; ESI-MS(−) m/z 560.6 (M−H); ¹H NMR (400 MHz,CHLOROFORM-d) δ 7.36 (m, 5H), 6.37 (m, 1H), 5.18 (s, 2H), 4.67 (m, 1H),2.40 (m, 1H), 2.21 (m, 6H), 2.02 (m, 1H), 1.61 (m, 6H), 1.45 (s, 9H),1.40-1.16 (m, 18H).

Step 3

To a 50 ml round bottom flask was added(S)-5-(benzyloxy)-4-(16-(tert-butoxy)-16-oxohexadecanamido)-5-oxopentanoicacid (836 mg, 1.488 mmol), CH₂Cl₂ (8 mL),1-(9-Fluorenylmethyloxycarbonyl-amino)-4,7,10-trioxa-13-tridecanaminehydrochloride (713 mg, 1.488 mmol), Hunig's Base (0.780 mL, 4.46 mmol)and HATU (736 mg, 1.935 mmol). The flask was sealed with a septum andkept under a blanket of nitrogen and stirred overnight at rt. The nextday the reaction solvent was evaporated in vacuo. The crude product waspurified on silica gel chromatography eluting with 20% Acetone/80%Hexanes to 60% Acetone/40% hexanes. The pure fractions were combined andevaporated in vacuo affording (S)-tert-butyl22-((benzyloxy)carbonyl)-1-(9H-fluoren-9-yl)-3,19,24-trioxo-2,8,11,14-tetraoxa-4,18,23-triazanonatriacontan-39-oate(520 mg, 0.527 mmol, 35.4% yield). Analysis LCMS Condition A: Retentiontime=6.096 min; ESI-MS(+) m/z 987.0 (M+H); ¹H NMR (400 MHz,CHLOROFORM-d) δ 7.79 (d, J=8.0 Hz, 2H), 7.63 (d, J=8.0 Hz, 2H), 7.42 (t,J=8.0 Hz, 2H), 7.38-7.30 (m, 7H), 6.83 (m, 1H), 6.53 (m, 1H), 5.49 (m,1H), 5.18 (m, 2H), 4.58 (m, 1H), 4.42 (d, J=4.0 Hz, 2H), 4.23 (m, 1H),3.63 (m, 6H), 3.55 (m, 6H), 3.33 (m, 4H), 2.22 (m, 7H), 2.02 (m, 1H),1.77 (m, 4H), 1.63 (m, 4H), 1.47 (s, 9H), 1.37-1.20 (m, 20H).

Step 4

To a 25 ml round bottom flask was added(S)-tert-butyl22-((benzyloxy)carbonyl)-1-(9H-fluoren-9-yl)-3,19,24-trioxo-2,8,11,14-tetraoxa-4,18,23-triazanonatriacontan-39-oate(520 mg, 0.527 mmol), Methanol (10 mL), and PALLADIUM ON CARBON (56.1mg, 0.053 mmol). The flask was sealed with a septum and charged withHYDROGEN (1.063 mg, 0.527 mmol) via a balloon. The next day the reactionwas checked by LC/MS and was complete. The reaction was filtered throughcelite to remove the catalyst and the filtrate was evaporated in vacuoaffording(S)-22-(16-(tert-butoxy)-16-oxohexadecanamido)-1-(9H-fluoren-9-yl)-3,19-dioxo-2,8,11,14-tetraoxa-4,18-diazatricosan-23-oicacid (415 mg, 0.463 mmol, 88% yield). This material was used as iswithout purification. Analysis LCMS Condition A: Retention time=4.535min; ESI-MS(−) m/z 895.0 (M−H).

Step 5: Modified Chlorotrityl Resin V

To a peptide vessel was added 2-chlorotrityl resin (926 mg, 1.482 mmol),(S)-22-(16-(tert-butoxy)-16-oxohexadecanamido)-1-(9H-fluoren-9-yl)-3,19-dioxo-2,8,11,14-tetraoxa-4,18-diazatricosan-23-oicacid (415 mg, 0.463 mmol), CH₂Cl₂ (8 mL), 1-chloro-4-methylbenzene (58.6mg, 0.463 mmol), and N-ethyl-N-isopropylpropan-2-amine (0.565 mL, 3.24mmol). The vessel was sealed and shaken on a wrist action shaker for 30min. The reaction was complete by analyzing the LC/MS and comparing theratio of the internal standard 1-chloro-4-methylbenzene (58.6 mg, 0.463mmol) vs. starting acid. The resin was then diluted with 20 ml of a 9:1Methanol/Hunigs base solution and quickly filtered and washed with DMF3×, CH₂Cl₂ 3× and finally diethyl ether. The resin was dried in vacuoand used as is for peptide synthesis with an assumed loading of 0.5meq/g.

Preparation of Example 11158

Example 11158 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin V was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 42 mg, and its estimated purity by LCMSanalysis was 99%. Analysis LCMS Condition A: Retention time=3.710 min;ESI-MS(+) m/z 1302.1958 (M+2H).

Preparation of Example 11159

Example 11159 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin V was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 30 mg, and its estimated purity by LCMSanalysis was 97.9%. Analysis LCMS Condition A: Retention time=3.701 min;ESI-MS(+) m/z 1309.2045 (M+2H).

Preparation of Example 11160

Example 11160 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin M was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 23 mg, and its estimated purity by LCMSanalysis was 97.1%. Analysis LCMS Condition A: Retention time=4.636 min;ESI-MS(+) m/z 1316.2122 (M+2H).

Preparation of Example 11161

Example 11161 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin V was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 40 mg, and its estimated purity by LCMSanalysis was 97.9%. Analysis LCMS Condition A: Retention time=4.161 min;ESI-MS(+) m/z 1216.1731 (M+2H).

Preparation of Example 11162

Example 11162 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin V was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 22 mg, and its estimated purity by LCMSanalysis was 96.3%. Analysis LCMS Condition A: Retention time=3.825 min.

Preparation of Example 11163

Example 11163 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin M was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 11 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition A: Retention time=4.066 min;ESI-MS(+) m/z 1323.2176 (M+2H).

Preparation of Example 11164

Example 11164 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin U was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 7 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition A: Retention time=3.668 min;ESI-MS(+) m/z 1347.2103 (M+2H).

Preparation of Example 11165

Example 11165 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin U was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 28 mg, and its estimated purity by LCMSanalysis was 94.8%. Analysis LCMS Condition A: Retention time=3.653 min;ESI-MS(+) m/z 1354.2194 (M+2H).

Preparation of Example 11166

Example 11166 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin M was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 12 mg, and its estimated purity by LCMSanalysis was 93.1%. Analysis LCMS Condition A: Retention time=3.690 min;ESI-MS(+) m/z 1316.2126 (M+2H).

Preparation of Example 11167

Example 11167 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin M was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 24 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition A: Retention time=3.811 min;ESI-MS(+) m/z 1323.2204 (M+2H).

Preparation of Example 11168

Example 11168 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin V was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 34 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition A: Retention time=3.798 min;ESI-MS(+) m/z 1244.6830 (M+2H).

Preparation of Example 11169

Example 11169 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin V was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 29 mg, and its estimated purity by LCMSanalysis was 96%. Analysis LCMS Condition A: Retention time=3.423 min;ESI-MS(+) m/z 1359.7108 (M+2H).

Preparation of Example 11170

Example 11170 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin V was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 32 mg, and its estimated purity by LCMSanalysis was 96.3%. Analysis LCMS Condition A: Retention time=3.426 min;ESI-MS(+) m/z 1366.7180 (M+2H).

Preparation of Example 11171

Example 11171 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin V was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 35 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition A: Retention time=4.001 min;ESI-MS(+) m/z 1251.6929 (M+2H).

Preparation of modified chlorotrityl chloride resin W

Step 1

To a 50 ml round bottom flask was added(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-aminopropanoic acid,HCl (500 mg, 1.378 mmol), N,N-Dimethylformamide (12 mL), 1-tert-butyl18-(perfluorophenyl) octadecanedioate (1109 mg, 2.067 mmol), and Hunig'sBase (0.963 mL, 5.51 mmol). The flask was sealed with a septum and keptunder a blanket of nitrogen and stirred overnight at rt. The next daythe reaction was poured into a saturated citric acid solution andextracted with CH₂Cl₂ 3×. The organic layers were combined and washedwith brine, dried over Na₂SO₄ and evaporated in vacuo. The crude productwas purified on silica gel chromatography eluting with 100% CH₂Cl₂ then5% MeOH in 95% CH₂Cl₂. The pure fractions were combined and evaporatedin vacuo affording(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)propanoicacid (774 mg, 1.140 mmol, 83% yield). Analysis LCMS Condition A:Retention time=3.698 min; ESI-MS(−) m/z 677.7 (M−H); ¹H NMR (400 MHz,CHLOROFORM-d) δ 7.78 (d, J=7.5 Hz, 2H), 7.59 (d, J=7.5 Hz, 2H), 7.42 (t,J=7.4 Hz, 2H), 7.33 (t, J=7.2 Hz, 2H), 7.26 (d, J=5.0 Hz, 1H), 5.67 (s,1H), 4.59-4.49 (m, 1H), 4.41 (d, J=7.0 Hz, 2H), 4.23 (t, J=7.2 Hz, 1H),3.83-3.69 (m, 1H), 3.67-3.55 (m, 1H), 2.32-2.16 (m, 4H), 1.70-1.53 (m,4H), 1.46 (s, 9H), 1.36-1.17 (m, 24H).

Step 2

To a peptide vessel was added 2-Chlorotrityl resin (2280 mg, 3.65 mmol),(S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)propanoicacid (774 mg, 1.140 mmol), CH₂Cl₂ (16 mL), 1-chloro-4-methylbenzene(43.3 mg, 0.342 mmol), and N-ethyl-N-isopropylpropan-2-amine (1.390 mL,7.98 mmol). The vessel was sealed and shaken on a wrist action shakerfor 30 min. The reaction was complete by analyzing the LC/MS andcomparing the ratio of the internal standard 1-chloro-4-methylbenzene(43.3 mg, 0.342 mmol) vs. starting acid. The resin was then diluted with20 ml of a 9:1 Methanol/Hunigs base solution and quickly filtered andwashed with DMF 3×, CH₂Cl₂ 3× and finally diethyl ether. The resin wasdried in vacuo and was used as is with an assumed loading of 0.5 meq/gfor the synthesis of the desired proteins.

Preparation of Example 11172

Example 11172 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 27 mg, and its estimated purity by LCMSanalysis was 93.5%. Analysis LCMS Condition A: Retention time=4.218 min;ESI-MS(+) m/z 1193.6278 (M+2H).

Preparation of Example 11173

Example 11173 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 35 mg, and its estimated purity by LCMSanalysis was 95.9%. Analysis LCMS Condition A: Retention time=4.218 min;ESI-MS(+) m/z 1200.6358 (M+2H).

Preparation of Example 11174

Example 11174 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin M was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 29 mg, and its estimated purity by LCMSanalysis was 95.3%. Analysis LCMS Condition A: Retention time=3.303 min;ESI-MS(+) m/z 1344.6838 (M+2H).

Preparation of Example 11175

Example 11175 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin M was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 19 mg, and its estimated purity by LCMSanalysis was 97.8%. Analysis LCMS Condition A: Retention time=3.71 min;ESI-MS(+) m/z 1316.7013 (M+2H).

Preparation of Example 11176

Example 11176 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin S was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 25 mg, and its estimated purity by LCMSanalysis was 97.5%. Analysis LCMS Condition A: Retention time=4.005 min;ESI-MS(+) m/z 1214.6502 (M+2H).

Preparation of Example 11177

Example 11177 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin S was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 27 mg, and its estimated purity by LCMSanalysis was 98.6%. Analysis LCMS Condition A: Retention time=4.010 min;ESI-MS(+) m/z 1221.6584 (M+2H).

Preparation of Example 11178

Example 11178 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin M was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 25 mg, and its estimated purity by LCMSanalysis was 96.7%. Analysis LCMS Condition A: Retention time=3.985 min;ESI-MS(+) m/z 1324.6806 (M+2H).

Preparation of Example 11179

Example 11179 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin M was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 30 mg, and its estimated purity by LCMSanalysis was 95.5%. Analysis LCMS Condition A: Retention time=3.928 min;ESI-MS(+) m/z 1350.1861 (M+2H).

Preparation of Example 11180

Example 11180 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 40 mg, and its estimated purity by LCMSanalysis was 97.7%. Analysis LCMS Condition A: Retention time=4.750 min;ESI-MS(+) m/z 1275.2005 (M+2H).

Preparation of Example 11181

Example 11181 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 25 mg, and its estimated purity by LCMSanalysis was 97.8%. Analysis LCMS Condition A: Retention time=4.383 min;ESI-MS(+) m/z 1361.2253 (M+2H).

Preparation of Example 11182

Example 11182 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 21 mg, and its estimated purity by LCMSanalysis was 94.9%. Analysis LCMS Condition A: Retention time=4.000 min.

Preparation of Example 11183

Example 11183 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 27 mg, and its estimated purity by LCMSanalysis was 97.0%. Analysis LCMS Condition A: Retention time=3.933 min.

Preparation of Example 11184

Example 11184 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin V was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 21 mg, and its estimated purity by LCMSanalysis was 96.0%. Analysis LCMS Condition A: Retention time=3.803 min.

Preparation of Example 11185

Example 11185 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 25 mg, and its estimated purity by LCMSanalysis was 94.5%. Analysis LCMS Condition A: Retention time=4.056 min.

Preparation of Example 11186

Example 11186 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin S was used in this synthesis. The crude material waspurified via preparative LC/MS with the following conditions: The crudematerial was purified via preparative LC with the following conditions:Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm. Mobile Phase A:10:90 acetonitrile: water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 30 mg, and its estimated purity by LCMSanalysis was 95%. Analysis LCMS Condition A: Retention time=4.163 min.

Preparation of Example 11187

Example 11187 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin A was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 25-75% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 15.5 mg, and itsestimated purity by LCMS analysis was 98%. Analysis LCMS Condition C:Retention time=2.01 min; ESI-MS(+) m/z 1050.2 (M+2H); ESI-HRMS (+) m/z:1050.0926 (M+2H).

Preparation of Example 11188

Example 11188 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin A was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 25-75% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 23.5 mg, and itsestimated purity by LCMS analysis was 98%. Analysis LCMS Condition A:Retention time=5.12 min; ESI-MS(+) m/z 1078.9 (M+2H); ESI-HRMS(+) m/z:1078.6024 (M+2H).

Preparation of Example 11189

Example 11189 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin A was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: waters CSH C-18, 19×200 mm, 5-μm particles; MobilePhase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; MobilePhase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid;Gradient: 20-60% B over 30 minutes, then a 5-minute hold at 100% B;Flow: 20 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 17mg, and its estimated purity by LCMS analysis was 100%. Analysis LCMSCondition C: Retention time=2.06 min; ESI-MS(+) m/z 1107.3 (M+2H);ESI-HRMS(+) m/z: 1107.1154 (M+2H).

Preparation of Example 11190

Example 11190 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin A was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 25-75% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 10.7 mg, and itsestimated purity by LCMS analysis was 100%. Analysis LCMS Condition C:Retention time=4.46 min; ESI-MS(+) m/z 1136.5 (M+2H); ESI-HRMS(+) m/z:1136.1179 (M+2H).

Preparation of Example 11191

Example 11191 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin B was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 30-70% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 6.4 mg, and itsestimated purity by LCMS analysis was 96%. Analysis LCMS Condition D:Retention time=2.54 min; ESI-MS(+) m/z 1064.0 (M+2H); ESI-HRMS(+) m/z:1064.1084 (M+2H).

Preparation of Example 11192

Example 11192 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin B was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 30-85% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 7.1 mg, and itsestimated purity by LCMS analysis was 100%. Analysis LCMS Condition C:Retention time=2.08 min; ESI-MS(+) m/z 1092.5 (M+2H); ESI-HRMS(+) m/z:1092.6200 (M+2H).

Preparation of Example 11193

Example 11205 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin B was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 30-70% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 8.3 mg, and itsestimated purity by LCMS analysis was 98%. Analysis LCMS Condition A:Retention time=5.14 min; ESI-MS(+) m/z 1121.4 (M+2H); ESI-HRMS(+) m/z:1121.1305 (M+2H).

Preparation of Example 11194

Example 11194 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin C was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 30-70% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 9.2 mg, and itsestimated purity by LCMS analysis was 100%. Analysis LCMS Condition C:Retention time=1.80 min; ESI-MS(+) m/z 1093.3 (M+2H); ESI-HRMS(+) m/z:1093.0963 (M+2H).

Preparation of Example 11195

Example 11195 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin C was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 25-65% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 7.7 mg, and itsestimated purity by LCMS analysis was 97%. Analysis LCMS Condition D:Retention time=1.82 min; ESI-MS(+) m/z 1122.4 (M+2H); ESI-HRMS(+) m/z:1122.0992 (M+2H).

Preparation of Example 11196

Example 11196 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin C was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 9.4 mg, and its estimated purity by LCMSanalysis was 97%.

Analysis LCMS Condition A: Retention time=4.84 min; ESI-MS(+) m/z 1036.3(M+2H); ESI-HRMS(+) m/z: 1036.0766 (M+2H).

Preparation of Example 11197

Example 11197 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin C was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 7.2 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition A: Retention time=4.73 min; ESI-MS(+) m/z 1064.7(M+2H); ESI-HRMS (+) m/z: 1064.5886 (M+2H).

Preparation of Example 11198

Example 11198 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin D was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-80% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 17.1 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition B: Retention time=2.25 min;ESI-MS(+) m/z 1162.7 (M+2H); ESI-HRMS(+) m/z: 1162.6110 (M+2H).

Preparation of Example 11199

Example 11199 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin D was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-80% B over 25 minutes,then a 5-minute hold at 100% dried via centrifugal evaporation. Theyield of the product was 12.8 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition B: Retention time=3.38 min; ESI-MS(+) m/z 1115.4(M+2H).

Preparation of Example 11200

Example 11200 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin D was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-85% B over 25 minutes,then a 5-minute hold at 100% dried via centrifugal evaporation. Theyield of the product was 20.5 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition B: Retention time=2.22 min; ESI-MS(+) m/z 1191.4(M+2H); ESI-HRMS(+) m/z: 1191.1218 (M+2H).

Preparation of Example 11201

Example 11201 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin D was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-80% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 19.2 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition B: Retention time=2.40 min; ESI-MS(+) m/z 1143.7(M+2H); ESI-HRMS(+) m/z: 1143.6036 (M+2H).

Preparation of Example 11202

Example 11202 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin D was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-80% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min.

Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 14 mg, and itsestimated purity by LCMS analysis was 98%. Analysis LCMS Condition B:Retention time=2.57 min; ESI-MS(+) m/z 1114.8 (M+2H); ESI-HRMS(+) m/z:1114.6003 (M+2H).

Preparation of Example 11203

Example 11203 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin D was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-85% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 19 mg, and its estimated purity by LCMSanalysis was 99%. Analysis LCMS Condition B: Retention time=2.44 min;ESI-MS(+) m/z 1162.4 (M+2H); ESI-HRMS(+) m/z: 1162.1192 (M+2H).

Preparation of Example 11204

Example 11204 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 20-60% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 16.7 mg, and itsestimated purity by LCMS analysis was 97%. Analysis LCMS Condition D:Retention time=1.94 min; ESI-MS(+) m/z 1022.4 (M+2H); ESI-HRMS(+) m/z:1022.0615 (M+2H).

Preparation of Example 11205

Example 11205 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 20-60% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 15.5 mg, and itsestimated purity by LCMS analysis was 95%. Analysis LCMS Condition D:Retention time=1.87 min; ESI-MS(+) m/z 1050.3 (M+2H); ESI-HRMS(+) m/z:1050.5731 (M+2H).

Preparation of Example 11206

Example 11206 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 50-90% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 15.5 mg, and itsestimated purity by LCMS analysis was 95%. Analysis LCMS Condition D:Retention time=1.84 min; ESI-MS(+) m/z 1079.1 (M+2H); ESI-HRMS(+) m/z:1079.0823 (M+2H).

Preparation of Example 11207

Example 11207 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% dried via centrifugal evaporation. Theyield of the product was 24.7 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition A: Retention time=4.14 min;ESI-MS(+) m/z 1098.2 (M+2H); ESI-HRMS(+) m/z: 1098.0917 (M+2H).

Preparation of Example 11208

Example 11208 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 6 mg, and itsestimated purity by LCMS analysis was 100%. Analysis LCMS Condition B:Retention time=2.41 min; ESI-MS(+) m/z 1108.5 (M+2H); ESI-HRMS(+) m/z:1108.0856 (M+2H).

Preparation of Example 11209

Example 11209 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-55% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 9.5 mg, and itsestimated purity by LCMS analysis was 100%. Analysis LCMS Condition D:Retention time=1.83 min; ESI-MS(+) m/z 1070.0 (M+2H); ESI-HRMS(+) m/z:1069.5802 (M+2H).

Preparation of Example 11210

Example 11210 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-55% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 3.7 mg, and itsestimated purity by LCMS analysis was 100%. Analysis LCMS Condition A:Retention time=3.96 min; ESI-MS(+) m/z 1126.8 (M+2H); ESI-HRMS(+) m/z:1126.6015 (M+2H).

Preparation of Example 11211

Example 11211 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-55% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 7.6 mg, and itsestimated purity by LCMS analysis was 96%. Analysis LCMS Condition C:Retention time=1.46 min; ESI-MS(+) m/z 1155.2 (M+2H); ESI-HRMS(+) m/z:1155.6043 (M+2H).

Preparation of Example 11212

Example 11212 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-75% B over 25 minutes,then a 5-minute hold at 100% dried via centrifugal evaporation. Theyield of the product was 18 mg, and its estimated purity by LCMSanalysis was 97%. Analysis LCMS Condition A: Retention time=3.97 min;ESI-MS(+) m/z 1108.2 (M+2H); ESI-HRMS(+) m/z: 1108.0828 (M+2H).

Preparation of Example 11213

Example 11213 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-75% B over 25 minutes,then a 5-minute hold at 100% dried via centrifugal evaporation. Theyield of the product was 14.4 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition A: Retention time=3.58 min;ESI-MS(+) m/z 1155.9 (M+2H); ESI-HRMS(+) m/z: 1155.6020 (M+2H).

Preparation of Example 11214

Example 11214 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 8.8 mg, and its estimated purity by LCMSanalysis was 99%. Analysis LCMS Condition A: Retention time=4.34 min;ESI-MS(+) m/z 1173.3 (M+2H); ESI-HRMS(+) m/z: 1173.1503 (M+2H).

Preparation of Example 11215

Example 11215 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 13 mg, and its estimated purity by LCMSanalysis was 99%. Analysis LCMS Condition A: Retention time=4.02 min;ESI-MS(+) m/z 1108.3 (M+2H); ESI-HRMS(+) m/z: 1107.5930 (M+2H).

Preparation of Example 11216

Example 11216 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 17.7 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition A: Retention time=3.72 min; ESI-MS(+) m/z 1155.3(M+2H); ESI-HRMS(+) m/z: 1155.1126 (M+2H).

Preparation of Example 11217

Example 11217 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 17.9 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition A: Retention time=3.55 min;ESI-MS(+) m/z 1165.6 (M+2H); ESI-HRMS(+) m/z: 1165.5995 (M+2H).

Preparation of Example 11218

Example 11218 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-80% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 17.7 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition A: Retention time=3.17 min;ESI-MS(+) m/z 1213.1 (M+2H); ESI-HRMS(+) m/z: 1213.1169 (M+2H).

Preparation of Example 11219

Example 11219 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 21.7 mg, and its estimated purity by LCMSanalysis was 96%. Analysis LCMS Condition A: Retention time=3.83 min;ESI-MS(+) m/z 1220.8 (M+2H); ESI-HRMS(+) m/z: 1220.6713 (M+2H).

Preparation of Example 11220

Example 11220 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-80% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 17.3 mg, and its estimated purity by LCMSanalysis was 99%. Analysis LCMS Condition A: Retention time=3.49 min;ESI-MS(+) m/z 1184.2 (M+2H); ESI-HRMS(+) m/z: 1184.1140 (M+2H).

Preparation of Example 11221

Example 11221 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-80% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 15.8 mg, and its estimated purity by LCMSanalysis was 99%. Analysis LCMS Condition A: Retention time=3.79 min;ESI-MS(+) m/z 1136.6 (M+2H); ESI-HRMS(+) m/z: 1136.5948 (M+2H).

Preparation of Example 11222

Example 11222 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-80% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 23.7 mg, and its estimated purity by LCMSanalysis was 97%.

Analysis LCMS Condition A: Retention time=3.47 min; ESI-MS(+) m/z 1184.3(M+2H); ESI-HRMS(+) m/z: 1184.1132 (M+2H).

Preparation of Example 11223

Example 11223 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetyl chloride coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin E was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-80% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 24.8 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition A: Retention time=3.79 min;ESI-MS(+) m/z 1136.8 (M+2H); ESI-HRMS(+) m/z: 1136.5948 (M+2H).

Preparation of Example 11224

Example 11224 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin F was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 14 mg, and its estimated purity by LCMSanalysis was 99%. Analysis LCMS Condition B: Retention time=2.13 min;ESI-MS(+) m/z 1317.0 (M+2H); ESI-HRMS(+) m/z: 1316.2122 (M+2H).

Preparation of Example 11225

Example 11225 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin F was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 35.3 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition B: Retention time=2.27 min;ESI-MS(+) m/z 1374.2 (M+2H); ESI-HRMS(+) m/z: 1373.7259 (M+2H).

Preparation of Example 11226

Example 11226 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin F was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 18.7 mg, and its estimated purity by LCMSanalysis was 100%. Analysis LCMS Condition B: Retention time=2.54 min;ESI-MS(+) m/z 1337.5 (M+2H); ESI-HRMS(+) m/z: 1337.2332 (M+2H).

Preparation of Example 11227

Example 11227 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin F was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 24.7 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition B: Retention time=2.32 min; ESI-MS(+) m/z 1345.5(M+2H); ESI-HRMS(+) m/z: 1345.2147 (M+2H).

Preparation of Example 11228

Example 11228 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin F was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 24.7 mg, and its estimated purity by LCMSanalysis was 100%.

Analysis LCMS Condition B: Retention time=2.28 min; ESI-MS(+) m/z 1352.7(M+2H); ESI-HRMS(+) m/z: 1352.2227 (M+2H).

Preparation of Example 11229

Example 11229 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin F was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 14.1 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition B: Retention time=2.35 min; ESI-MS(+) m/z 1309.8(M+2H); ESI-HRMS(+) m/z: 1309.2043 (M+2H).

Preparation of Example 11230

Example 11230 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin F was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 23.4 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition B: Retention time=2.68 min; ESI-MS(+) m/z 1380.5(M+2H); ESI-HRMS(+) m/z: 1380.7350 (M+2H).

Preparation of Example 11231

Example 11231 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin F was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 19 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition B: Retention time=2.54 min;ESI-MS(+) m/z 1330.5 (M+2H); ESI-HRMS(+) m/z: 1330.2260 (M+2H).

Preparation of Example 11232

Example 11232 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin G was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 23.4 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition B: Retention time=2.28 min;ESI-MS(+) m/z 1367.1 (M+2H); ESI-HRMS(+) m/z: 1366.7180 (M+2H).

Preparation of Example 11233

Example 11233 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin G was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 29.4 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition B: Retention time=2.18 min;ESI-MS(+) m/z 1345.3 (M+2H); ESI-HRMS(+) m/z: 1345.2151 (M+2H).

Preparation of Example 11234

Example 11234 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin G was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-75% B over 25 minutes,then a 5-minute hold at 100% dried via centrifugal evaporation. Theyield of the product was 19.7 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition B: Retention time=2.35 min; ESI-MS(+) m/z 1317.1(M+2H); ESI-HRMS(+) m/z: 1316.2133 (M+2H).

Preparation of Example 11235

Example 11235 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin G was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-75% B over 25 minutes,then a 5-minute hold at 100% dried via centrifugal evaporation. Theyield of the product was 18.4 mg, and its estimated purity by LCMSanalysis was 99%. Analysis LCMS Condition B: Retention time=2.26 min;ESI-MS(+) m/z 1374.1 (M+2H); ESI-HRMS(+) m/z: 1373.7263 (M+2H).

Preparation of Example 11236

Example 11236 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin G was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-75% B over 25 minutes,then a 5-minute hold at 100% dried via centrifugal evaporation. Theyield of the product was 21 mg, and its estimated purity by LCMSanalysis was 97%.

Analysis LCMS Condition B: Retention time=2.31 min; ESI-MS(+) m/z 1338.1(M+2H); ESI-HRMS(+) m/z: 1338.2058 (M+2H).

Preparation of Example 11237

Example 11237 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin G was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 14.7 mg, and its estimated purity by LCMSanalysis was 97%.

Analysis LCMS Condition B: Retention time=2.45 min; ESI-MS(+) m/z 1309.3(M+2H); ESI-HRMS(+) m/z: 1309.2068 (M+2H).

Preparation of Example 11238

Example 11238 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin H was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-80% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 27.7 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition B: Retention time=2.37 min; ESI-MS(+) m/z 1295.5(M+2H); ESI-HRMS(+) m/z: 1295.1866 (M+2H).

Preparation of Example 11239

Example 11239 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin H was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-80% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 21.4 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition B: Retention time=2.42 min; ESI-MS(+) m/z 1295.6(M+2H); ESI-HRMS(+) m/z: 1295.1864 (M+2H).

Preparation of Example 11240

Example 11240 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin H was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 25.7 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition B: Retention time=2.35 min; ESI-MS(+) m/z 1353.1(M+2H); ESI-HRMS(+) m/z: 1352.7005 (M+2H).

Preparation of Example 11241

Example 11241 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin H was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 18.4 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition B: Retention time=2.31 min; ESI-MS(+) m/z 1360.0(M+2H); ESI-HRMS(+) m/z: 1359.7091 (M+2H).

Preparation of Example 11242

Example 11242 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin H was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-80% B over 25 minutes,then a 5-minute hold at 100% dried via centrifugal evaporation. Theyield of the product was 27.4 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition B: Retention time=2.54 min; ESI-MS(+) m/z 1209.3(M+2H); ESI-HRMS(+) m/z: 1209.1651 (M+2H).

Preparation of Example 11243

Example 11243 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin H was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 21.7 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition B: Retention time=2.34 min; ESI-MS(+) m/z 1302.5(M+2H); ESI-HRMS (+) m/z: 1302.1978 (M+2H).

Preparation of Example 11244

Example 11244 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin H was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 33.7 mg, and its estimated purity by LCMSanalysis was 96%. Analysis LCMS Condition B: Retention time=2.14 min;ESI-MS(+) m/z 1324.3 (M+2H); ESI-HRMS(+) m/z: 1324.1926 (M+2H).

Preparation of Example 11245

Example 11245 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin H was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 26.3 mg, and its estimated purity by LCMSanalysis was 99%. Analysis LCMS Condition B: Retention time=2.15 min;ESI-MS(+) m/z 1331.5 (M+2H); ESI-HRMS(+) m/z: 1331.2001 (M+2H).

Preparation of Example 11246

Example 11246 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin I was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 12.1 mg, and its estimated purity by LCMSanalysis was 99%.

Analysis LCMS Condition B: Retention time=2.59 min; ESI-MS(+) m/z 1337.4(M+2H); ESI-HRMS(+) m/z: 1337.2369 (M+2H).

Preparation of Example 11247

Example 11247 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin I was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 12.8 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition B: Retention time=2.61 min;ESI-MS(+) m/z 1330.4 (M+2H); ESI-HRMS(+) m/z: 1330.2305 (M+2H).

Preparation of Example 11248

Example 11248 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin I was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 15-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 4.4 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition B: Retention time=2.79 min;ESI-MS(+) m/z 1244.5 (M+2H); ESI-HRMS(+) m/z: 1244.2027 (M+2H).

Preparation of Example 11249

Example 11249 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin I was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 30 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 5.9 mg, and its estimated purity by LCMSanalysis was 97%. Analysis LCMS Condition B: Retention time=2.49 min;ESI-MS(+) m/z 1395.2.

Preparation of Example 11250

Example 11250 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin I was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 30 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 12.4 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition B: Retention time=2.49 min;ESI-MS(+) m/z 1388.2.

Preparation of Example 11251

Example 11251 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin I was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 30 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 17 mg, and its estimated purity by LCMSanalysis was 98%.

Analysis LCMS Condition B: Retention time=2.47 min; ESI-MS(+) m/z1366.7.

Preparation of Example 11252

Example 11252 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin I was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 30 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 20-60% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 18 mg, and its estimated purity by LCMSanalysis was 97%.

Analysis LCMS Condition B: Retention time=2.47 min; ESI-MS(+) m/z1359.7.

Preparation of Example 11253

Example 11253 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin J was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 30 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 25-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 18.5 mg, and its estimated purity by LCMSanalysis was 95%. Analysis LCMS Condition B: Retention time=2.23 min;ESI-MS(+) m/z 1295.4.

Preparation of Example 11254

Example 11254 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin J was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 30 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 25-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 30.6 mg, and its estimated purity by LCMSanalysis was 98%. Analysis LCMS Condition B: Retention time=2.21 min;ESI-MS(+) m/z 1288.5.

Preparation of Example 11255

Example 11255 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin J was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 30 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 25-70% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 9.7 mg, and its estimated purity by LCMSanalysis was 96%. Analysis LCMS Condition B: Retention time=2.20 min;ESI-MS(+) m/z 1295.4.

Preparation of Example 11256

Example 11256 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin J was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 30 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 25-70% B over 25 minutes,then a 5-minute hold at 100% dried via centrifugal evaporation. Theyield of the product was 21.5 mg, and its estimated purity by LCMSanalysis was 97%. Analysis LCMS Condition B: Retention time=2.20 min;ESI-MS(+) m/z 1288.5.

Preparation of Example 11257

Example 11257 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin N was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 22.1 mg, and its estimated purity by LCMSanalysis was 97%.Analysis LCMS Condition B: Retention time=2.38 min; ESI-MS(+) m/z 1316.7(M+2H); ESI-HRMS(+) m/z: 1316.2101 (M+2H).

Preparation of Example 11258

Example 11258 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin N was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 10-75% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 23.1 mg, and its estimated purity by LCMSanalysis was 98%.Analysis LCMS Condition B: Retention time=2.66 min; ESI-MS(+) m/z 1223.4(M+2H); ESI-HRMS(+) m/z: 1223.1770 (M+2H).

Preparation of Example 11259

Example 11259 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin Q was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 30 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 25-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 4.1 mg, and its estimated purity by LCMSanalysis was 98%.Analysis LCMS Condition B: Retention time=2.40 min; ESI-MS(+) m/z1202.5.

Preparation of Example 11260

Example 11260 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin Q was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 30 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 25-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 12.2 mg, and its estimated purity by LCMSanalysis was 99%.Analysis LCMS Condition B: Retention time=2.34 min; ESI-MS(+) m/z 1231.1

Preparation of Example 11261

Example 11261 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure B”, “Symphony Method A:Secondary amine-coupling procedure B”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modified2-chlorotrityl chloride resin Q was used in this synthesis. The crudematerial was purified via preparative LC/MS with the followingconditions: Column: XSelect CSH Prep C18, 5-μm OBD, 30 mm×250 mm; MobilePhase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile Phase B: 90:10acetonitrile:water with 0.1% TFA; Gradient: 25-65% B over 25 minutes,then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containingthe desired product were combined and dried via centrifugal evaporation.The yield of the product was 17.0 mg, and its estimated purity by LCMSanalysis was 97%.Analysis LCMS Condition B: Retention time=2.11 min; ESI-MS(+) m/z 1324.7

Preparation of Example 11262

Example 11262 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis.

The crude material was purified via preparative LC/MS with the followingconditions: The crude material was purified via preparative LC with thefollowing conditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250mm. Mobile Phase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile PhaseB: 90:10 acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 21 mg, and its estimatedpurity by LCMS analysis was 90.9%.

Analysis LCMS Condition A: Retention time=3.83 min; ESI-HRMS(+) m/z:1329.7089 (M+2H)

Preparation of Example 11263

Example 11263 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis.

The crude material was purified via preparative LC/MS with the followingconditions: The crude material was purified via preparative LC with thefollowing conditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250mm. Mobile Phase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile PhaseB: 90:10 acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 19 mg, and its estimatedpurity by LCMS analysis was 95.7%.

Analysis LCMS Condition A: Retention time=4.17 min; ESI-HRMS(+) m/z:1345.6943 (M+2H)

Preparation of Example 11264

Example 11264 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis.

The crude material was purified via preparative LC/MS with the followingconditions: The crude material was purified via preparative LC with thefollowing conditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250mm. Mobile Phase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile PhaseB: 90:10 acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 21 mg, and its estimatedpurity by LCMS analysis was 97.3%.

Analysis LCMS Condition A: Retention time=4.23 min; ESI-HRMS(+) m/z:1338.6835 (M+2H).

Preparation of Example 11265

Example 11265 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis.

The crude material was purified via preparative LC/MS with the followingconditions: The crude material was purified via preparative LC with thefollowing conditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250mm. Mobile Phase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile PhaseB: 90:10 acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 23 mg, and its estimatedpurity by LCMS analysis was 98.4%.

Analysis LCMS Condition A: Retention time=4.23 min; ESI-HRMS(+) m/z:1345.6946 (M+2H)

Preparation of Example 11266

Example 11266 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis.

The crude material was purified via preparative LC/MS with the followingconditions: The crude material was purified via preparative LC with thefollowing conditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250mm. Mobile Phase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile PhaseB: 90:10 acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 24 mg, and its estimatedpurity by LCMS analysis was 98.0%.

Analysis LCMS Condition A: Retention time=4.19 min.

Preparation of Example 11267

Example 11267 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001 except that the reactionwas run on 0.6 mmol scale, composed of the following general procedures:“Symphony Method A: Resin-swelling procedure”, “Symphony Method A:Standard-coupling procedure”, “Symphony Method A: Secondaryamine-coupling procedure A”, “Custom amino acids-coupling procedure”,“Chloroacetic acid coupling procedure A”, “Global Deprotection MethodA”, and “Cyclization Method A”. Modified chlorotrityl resin W was usedin this synthesis.

The crude material was purified via preparative LC/MS with the followingconditions: The crude material was purified via preparative LC with thefollowing conditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250mm. Mobile Phase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile PhaseB: 90:10 acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 280 mg, and its estimatedpurity by LCMS analysis was 95.6%.

Analysis LCMS Condition A: Retention time=3.98 min; ESI-HRMS(+) m/z:1370.7382 (M+2H).

Preparation of Example 11268

Example 11268 was prepared as follows. To a 1 dram vial was addedExample 11267 (30 mg, 10.11 μmol) in Water (0.5 mL). To this was addedt-BuOH (0.5 mL) and3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine (6.35mg, 0.020 mmol). The solution was stirred and then SODIUM ASCORBATE(2.60 mg, 0.013 mmol) and a solution of copper(II) sulfate pentahydrate(0.015 mL, 3.03 μmol) was added. The reaction was allowed to stir at rtfor 2 hr. The reaction was checked by LC/MS and was complete. The crudereaction mixture was injected directly onto a reverse phasechromatography column.

The crude material was purified via preparative LC/MS with the followingconditions: The crude material was purified via preparative LC with thefollowing conditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250mm. Mobile Phase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile PhaseB: 90:10 acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 16.3 mg, and its estimatedpurity by LCMS analysis was 98.7%.

Analysis LCMS Condition A: Retention time=4.01 min; ESI-HRMS(+) m/z:1528.6 (M+2H)

Preparation of Example 11269

Example 11269 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001, composed of the followinggeneral procedures: “Symphony Method A: Resin-swelling procedure”,“Symphony Method A: Standard-coupling procedure”, “Symphony Method A:Secondary amine-coupling procedure A”, “Custom amino acids-couplingprocedure”, “Chloroacetic acid coupling procedure A”, “GlobalDeprotection Method A”, and “Cyclization Method A”. Modifiedchlorotrityl resin W was used in this synthesis.

The crude material was purified via preparative LC/MS with the followingconditions: The crude material was purified via preparative LC with thefollowing conditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250mm. Mobile Phase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile PhaseB: 90:10 acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 21 mg, and its estimatedpurity by LCMS analysis was 90.6%.

Analysis LCMS Condition A: Retention time=4.25 min; ESI-HRMS(+) m/z:1330.7059 (M+2H).

Preparation of Example 11270

Example 11270 was prepared following the general synthetic sequencedescribed for the preparation of Example 0001 except that it was run on0.8 mmol scale and composed of the following general procedures:“Symphony Method A: Resin-swelling procedure”, “Symphony Method A:Standard-coupling procedure”, “Symphony Method A: Secondaryamine-coupling procedure A”, “Custom amino acids-coupling procedure”,“Chloroacetic acid coupling procedure A”, “Global Deprotection MethodA”, and “Cyclization Method A”. Modified chlorotrityl resin W was usedin this synthesis.

The crude material was purified via preparative LC/MS with the followingconditions: The crude material was purified via preparative LC with thefollowing conditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250mm. Mobile Phase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile PhaseB: 90:10 acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 140 mg, and its estimatedpurity by LCMS analysis was 97.4%.

Analysis LCMS Condition A: Retention time=4.07 min; ESI-HRMS(+) m/z:1363.7277 (M+2H).

Preparation of Example 11271

Example 11271 was prepared as follows. To a 1 dram vial was addedExample 11270 (40 mg, 0.015 mmol) in Water (0.5 mL). To this was addedt-BuOH (0.5 mL) and3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine (9.22mg, 0.029 mmol). The solution was stirred and then SODIUM ASCORBATE(3.78 mg, 0.019 mmol) and a solution of copper(II) sulfate pentahydrate(0.022 mL, 4.40 μmol) was added. The reaction was allowed to stir at rtfor 2 hr. The reaction was checked by LC/MS and was complete. The crudereaction mixture was injected directly onto a reverse phasechromatography column.

The crude material was purified via preparative LC/MS with the followingconditions: The crude material was purified via preparative LC with thefollowing conditions: Column: XSelect CSH Prep C18, 5-μm OBD, 19 mm×250mm. Mobile Phase A: 10:90 acetonitrile:water with 0.1% TFA; Mobile PhaseB: 90:10 acetonitrile:water with 0.1% TFA; Gradient: 20-65% B over 25minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 17.0 mg, and its estimatedpurity by LCMS analysis was 93.0%.

Analysis LCMS Condition A: Retention time=4.24 min; ESI-HRMS(+) m/z:1520.7970 (M+2H).

Analytical Data:

Mass Spectrometry: “ESI-MS(+)” signifies electrospray ionization massspectrometry performed in positive ion mode; “ESI-MS(−)” signifieselectrospray ionization mass spectrometry performed in negative ionmode; “ESI-HRMS(+)” signifies high-resolution electrospray ionizationmass spectrometry performed in positive ion mode; “ESI-HRMS(−)”signifies high-resolution electrospray ionization mass spectrometryperformed in negative ion mode. The detected masses are reportedfollowing the “m/z” unit designation. Compounds with exact massesgreater than 1000 were often detected as double-charged ortriple-charged ions.

Analysis Condition A:

Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50°C.; Gradient: 0% B, 0-100% B over 3 minutes, then a 0.5-minute hold at100% B; Flow: 1 mL/min; Detection: UV at 220 nm.

Analysis Condition B:

Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 methanol:water with 10 mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10 mM ammonium acetate; Temperature: 50° C.;Gradient: 0% B, 0-100% B over 3 minutes, then a 0.5-minute hold at 100%B; Flow: 0.5 mL/min; Detection: UV at 220 nm.

Analysis Condition C:

Column: Waters Aquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; MobilePhase A: 100% Water: 0.05% TFA; Mobile Phase B: 100% Acetonitrile: 0.05%TFA; Temperature: 40° C.; Gradient: 2-98% B over 1.5 minutes, then a0.5-minute hold at 100% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

Analysis Condition D:

Column: PHENOMENEX-LUNA 2.0×30 mm 3 um; Mobile Phase A: 90% Water—10%Methanol—0.1% TFA; Mobile Phase B: 10% Water—90% Methanol—0.1% TFA;Gradient: 0-100% B over 2 minutes, then a 1 to 4 minute hold at 100% B;Flow: 1 mL/min; Detection: UV at 220 nm.

Analysis Condition E:

Column: Xbridge Phenyl, 3.0×150 mm, 3.5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10 mM ammonium acetate; Gradient: 5-100% Bover 15 minutes; Flow: 0.5 mL/min; Detection: UV at 220 nm.

Analysis Condition F:

Column: XBridge C18, 3.0×150 mm, 3.5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10 mM ammonium acetate; Gradient: 10-100% B over30 minutes; Flow: 0.5 mL/min; Detection: UV at 220 nm.

Analysis Condition G:

Column: Waters CSH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 acetonitrile:water with 0.1% TFA; Mobile Phase B: 95:5acetonitrile:water with 0.1% TFA; Temperature: 50° C.; Gradient: 0% B,0-100% B over 3 minutes, then a 0.5-minute hold at 100% B; Flow: 1mL/min; Detection: UV at 220 nm.

Analysis Condition H:

Column: Xbridge C18, 3.0×150 mm, 3.5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10 mM ammonium acetate; Gradient: 10-100% B over18 minutes; Flow: 0.5 mL/min; Detection: UV at 220 nm.

Analysis Condition I:

Column: XSelectCSH C18, 3.0×150 mm, 3.5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 0.1% TFA; Mobile Phase B: 95:5acetonitrile:water with 0.1% TFA; Gradient: 10-100% B over 15 minutes;Flow: 1.0 mL/min; Detection: UV at 220 nm.

Analysis Condition J:

Column: Zorbax Bonus RP, 3.0×150 mm, 3.5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 0.1% TFA; Mobile Phase B: 95:5acetonitrile:water with 0.1% TFA; Gradient: 10-100% B over 15 minutes;Flow: 1.0 mL/min; Detection: UV at 220 nm.

Analysis Condition K:

Column: Waters Aquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; MobilePhase A: 100% Water: 0.05% TFA; Mobile Phase B: 100% Acetonitrile: 0.05%TFA; Temperature: 50° C.; Gradient: 2-98% B over 3.0 minutes, then a0.5-minute hold at 100% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

General Procedures for Examples Intermediates 1300A-1400L

All manipulations were performed under automation on a Symphony-Xpeptide synthesizer (Protein Technologies). All procedures unless notedwere performed in a 10 mL polypropylene tube fitted with a bottom fit.The tube connects to the Prelude peptide synthesizer through both thebottom and the top of the tube. DMF and DCM can be added through the topof the tube, which washes down the sides of the tube equally. Theremaining reagents are added through the bottom of the tube and pass upthrough the frit to contact the resin. All solutions are removed throughthe bottom of the tube. “Periodic agitation” describes a brief pulse ofN₂ gas through the bottom frit; the pulse lasts approximately 5 secondsand occurs every 30 seconds. Chloroacetyl chloride solutions in DMF wereused within 24 h of preparation. Amino acid solutions were generally notused beyond three weeks from preparation. HATU solution was used within5 days of preparation. DMF=dimethylformamide;HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate; DIPEA=diisopropylethylamine;Rink=(2,4-dimethoxyphenyl)(4-alkoxyphenyl)methanamine, where “4-alkoxy”describes the position and type of connectivity to the polystyreneresin. The resin used is Merrifield polymer (polystyrene) with a Rinklinker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.56 mmol/gloading. Common amino acids used are listed below with side-chainprotecting groups indicated inside parenthesis. Fmoc-Ala-OH;Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH;Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH;Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH;Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH;Fmoc[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pra-OH;Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH;Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

For carboxamide products: the procedures describe an experimentperformed on a 0.100 mmol scale, where the scale is determined by theamount of Rink linker bound to the resin. This scale corresponds toapproximately 178 mg of the Rink-Merrifield resin described above. Priorto amino acid coupling, all peptide synthesis sequences began with aresin-swelling procedure, described below as “Resin-swelling procedure”.Coupling of amino acids to a primary amine N-terminus used the“Single-coupling procedure” described below. Coupling of amino acids toa secondary amine N-terminus used the “Double-coupling procedure”described below. Coupling of chloroacetylchloride to the N-terminus ofthe peptide is described by the “Chloroacetyl chloride couplingprocedure” detailed below.

Resin-Swelling Procedure:

To a 10 mL polypropylene solid-phase reaction vessel was addedMerrifield:Rink resin (178 mg, 0.100 mmol). The resin washed (swelled)three times as follows: to the reaction vessel was added DMF (2.0 mL),upon which the mixture was periodically agitated for 10 minutes beforethe solvent was drained through the frit.

Single-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperdine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitatedfor 3 minutes and then the solution was drained through the frit. To thereaction vessel was added piperdine:DMF (20:80 v/v, 2.0 mL). The mixturewas periodically agitated for 3 minutes and then the solution wasdrained through the frit. The resin washed successively six times asfollows: for each wash, DMF (2.0 mL) was added to top of the vessel (notthrough the bottom frit) and the resulting mixture was periodicallyagitated for 30 seconds before the solution was drained through thefrit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.0mL, 2 eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA(0.4M in DMF, 1.0 mL, 4 eq). The mixture was periodically agitated for15 minutes, then the reaction solution was drained through the frit. Theresin washed successively four times as follows: for each wash, DMF (2.0mL) was added to top of the vessel (not through the bottom frit) and theresulting mixture was periodically agitated for 30 seconds before thesolution was drained through the frit. To the reaction vessel was addedacetic anhydride (2.0 mL). The mixture was periodically agitated for 10minutes, then the solution was drained through the frit. The resinwashed successively four times as follows: for each wash, DMF (2.0 mL)was added to top of the vessel (not through the bottom frit) and theresulting mixture was periodically agitated for 90 seconds before thesolution was drained through the frit. The resulting resin was useddirectly in the next step.

Single-couplingfor-(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoic acid

The coupling was performed as above, only a 30 min agitation time wasused.

Double-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperdine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitatedfor 3 minutes and then the solution was drained through the frit. To thereaction vessel was added piperdine:DMF (20:80 v/v, 2.0 mL). The mixturewas periodically agitated for 3 minutes and then the solution wasdrained through the frit. The resin washed successively six times asfollows: for each wash, DMF (2.0 mL) was added to top of the vessel (notthrough the bottom frit) and the resulting mixture was periodicallyagitated for 30 seconds before the solution was drained through thefrit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.0mL, 2 eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA(0.4M in DMF, 1.0 mL, 4 eq). The mixture was periodically agitated for15 minutes, then the reaction solution was drained through the frit. Theresin was twice washed as follows: for each wash, DMF (2.0 mL) was addedto top of the vessel (not through the bottom fit) and the resultingmixture was periodically agitated for 30 seconds before the solution wasdrained through the frit. To the reaction vessel was added the aminoacid (0.2M in DMF, 1.0 mL, 2 eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq),and finally DIPEA (0.4M in DMF, 1.0 mL, 4 eq). The mixture wasperiodically agitated for 15 minutes, then the reaction solution wasdrained through the frit. The resin was twice washed as follows: foreach wash, DMF (2.0 mL) was added to top of the vessel (not through thebottom frit) and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added acetic anhydride (2.0 mL). The mixture wasperiodically agitated for 10 minutes, then the solution was drainedthrough the frit. The resin washed successively four times as follows:for each wash, DMF (2.0 mL) was added to top of the vessel (not throughthe bottom frit) and the resulting mixture was periodically agitated for90 seconds before the solution was drained through the frit. Theresulting resin was used directly in the next step.

Chloroacetyl Chloride Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperdine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitatedfor 3 minutes and then the solution was drained through the frit. To thereaction vessel was added piperdine:DMF (20:80 v/v, 2.0 mL). The mixturewas periodically agitated for 3 minutes and then the solution wasdrained through the frit. The resin washed successively six times asfollows: for each wash, DMF (2.0 mL) was added to top of the vessel (notthrough the bottom frit) and the resulting mixture was periodicallyagitated for 30 seconds before the solution was drained through thefrit. To the reaction vessel was added DIPEA (0.4M in DMF, 3.0 mL, 24eq), then chloroacetyl chloride (0.8M in DMF, 1.5 mL, 13.2 eq). Themixture was periodically agitated for 30 minutes, then the solution wasdrained through the frit. The resin was washed successively three timesas follows: for each wash, DMF (2.0 mL) was added to top of the vessel(not through the bottom frit) and the resulting mixture was periodicallyagitated for 90 seconds before the solution was drained through thefrit. The resin washed successively four times as follows: for eachwash, CH₂Cl₂ (2.0 mL) was added to top of the vessel (not through thebottom frit) and the resulting mixture was periodically agitated for 90seconds before the solution was drained through the frit. The resultingresin was placed under a N₂ stream for 15 minutes upon which the resinbecame rigid and easily handled.

For Carboxylic Acid C-Terminal Products:

The procedures describe an experiment performed on a 0.100 mmol scale,where the scale is determined by the amount of 2-chlorotrityl linkerbound to the resin. Commercial Fmoc-Gly-2-chlorotrityl resin was used,usually as a 0.92 meq/g loading. This scale corresponds to approximately109 mg of the Fmoc-Gly-2-chlorotrityl resin described above. Prior toamino acid coupling, all peptide synthesis sequences began with aresin-swelling procedure, described below as “Resin-swelling procedure”.Coupling of amino acids to a primary amine N-terminus used the“Single-coupling procedure” described below. Coupling of amino acids toa secondary amine N-terminus used the “Double-coupling procedure”described below. Coupling of chloroacetylchloride to the N-terminus ofthe peptide is described by the “Chloroacetyl chloride couplingprocedure” detailed below.

Resin-Swelling Procedure:

To a 10 mL polypropylene solid-phase reaction vessel was addedMerrifield:Rink resin (178 mg, 0.100 mmol). The resin washed (swelled)three times as follows: to the reaction vessel was added DMF (2.0 mL),upon which the mixture was periodically agitated for 10 minutes beforethe solvent was drained through the frit.

Single-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperdine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitatedfor 3 minutes and then the solution was drained through the frit. To thereaction vessel was added piperdine:DMF (20:80 v/v, 2.0 mL). The mixturewas periodically agitated for 3 minutes and then the solution wasdrained through the frit. The resin washed successively six times asfollows: for each wash, DMF (2.0 mL) was added to top of the vessel (notthrough the bottom frit) and the resulting mixture was periodicallyagitated for 30 seconds before the solution was drained through thefrit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.0mL, 2 eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA(0.4M in DMF, 1.0 mL, 4 eq). The mixture was periodically agitated for15 minutes, then the reaction solution was drained through the frit. Theresin washed successively four times as follows: for each wash, DMF (2.0mL) was added to top of the vessel (not through the bottom frit) and theresulting mixture was periodically agitated for 30 seconds before thesolution was drained through the frit. To the reaction vessel was addedDIPEA (0.4M in DMF, 1.0 mL, 4 eq), then acetic anhydride (2.0 mL). Themixture was periodically agitated for 10 minutes, then the solution wasdrained through the frit. The resin was washed successively four timesas follows: for each wash, DMF (2.0 mL) was added to top of the vessel(not through the bottom frit) and the resulting mixture was periodicallyagitated for 90 seconds before the solution was drained through thefrit. The resulting resin was used directly in the next step.

Single-couplingfor-(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoic acid

The coupling was performed as above, only a 30 min agitation time wasused.

Double-Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperdine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitatedfor 3 minutes and then the solution was drained through the frit. To thereaction vessel was added piperdine:DMF (20:80 v/v, 2.0 mL). The mixturewas periodically agitated for 3 minutes and then the solution wasdrained through the frit. The resin washed successively six times asfollows: for each wash, DMF (2.0 mL) was added to top of the vessel (notthrough the bottom frit) and the resulting mixture was periodicallyagitated for 30 seconds before the solution was drained through thefrit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.0mL, 2 eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq), and finally DIPEA(0.4M in DMF, 1.0 mL, 4 eq). The mixture was periodically agitated for15 minutes, then the reaction solution was drained through the frit. Theresin was twice washed as follows: for each wash, DMF (2.0 mL) was addedto top of the vessel (not through the bottom frit) and the resultingmixture was periodically agitated for 30 seconds before the solution wasdrained through the frit. To the reaction vessel was added the aminoacid (0.2M in DMF, 1.0 mL, 2 eq), then HATU (0.2M in DMF, 1.0 mL, 2 eq),and finally DIPEA (0.4M in DMF, 1.0 mL, 4 eq). The mixture wasperiodically agitated for 15 minutes, then the reaction solution wasdrained through the frit. The resin was twice washed as follows: foreach wash, DMF (2.0 mL) was added to top of the vessel (not through thebottom frit) and the resulting mixture was periodically agitated for 30seconds before the solution was drained through the frit. To thereaction vessel was added DIPEA (0.4M in DMF, 1.0 mL, 4 eq), then aceticanhydride (2.0 mL). The mixture was periodically agitated for 10minutes, then the solution was drained through the frit. The resinwashed successively four times as follows: for each wash, DMF (2.0 mL)was added to top of the vessel (not through the bottom frit) and theresulting mixture was periodically agitated for 90 seconds before thesolution was drained through the frit. The resulting resin was useddirectly in the next step.

Chloroacetyl Chloride Coupling Procedure:

To the reaction vessel containing resin from the previous step was addedpiperdine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitatedfor 3 minutes and then the solution was drained through the frit. To thereaction vessel was added piperdine:DMF (20:80 v/v, 2.0 mL). The mixturewas periodically agitated for 3 minutes and then the solution wasdrained through the frit. The resin washed successively six times asfollows: for each wash, DMF (2.0 mL) was added to top of the vessel (notthrough the bottom frit) and the resulting mixture was periodicallyagitated for 30 seconds before the solution was drained through thefrit. To the reaction vessel was added DIPEA (0.4M in DMF, 3.0 mL, 24eq), then chloroacetyl chloride (0.8M in DMF, 1.5 mL, 13.2 eq). Themixture was periodically agitated for 30 minutes, then the solution wasdrained through the frit. The resin washed successively three times asfollows: for each wash, DMF (2.0 mL) was added to top of the vessel (notthrough the bottom frit) and the resulting mixture was periodicallyagitated for 90 seconds before the solution was drained through thefrit. The resin washed successively four times as follows: for eachwash, CH₂Cl₂ (2.0 mL) was added to top of the vessel (not through thebottom frit) and the resulting mixture was periodically agitated for 90seconds before the solution was drained through the frit. The resultingresin was placed under a N₂ stream for 15 minutes upon which the resinbecame rigid and easily handled.

Global Deprotection Procedure:

A “deprotection solution” was prepared by combining in a 40 mL glassvial trifluoroacetic acid (22 mL), phenol (1.325 g), water (1.25 mL) andtriisopropylsilane (0.5 mL). The resin was removed from the reactionvessel and transferred to a 4 mL glass vial. To the vial was added the“deprotection solution” (2.0 mL). The mixture was vigorously mixed in ashaker (1000 RPM for 1 minute, then 500 RPM for 1.5 h). The mixture wasfiltered through a 0.2 micron syringe filter into a 18×150 mm test tube,and the solids were extracted with a second portion of the “deprotectionsolution” (1.0 mL). The combined filtrates, in the 18×150 mm test tube,were diluted with Et₂O (15 mL) upon which a significant amount of awhite solid precipitated. The mixture was centrifuged for 2 minutes,then the solution was decanted. The solids were suspended in Et₂O (20mL); the mixture was centrifuged for 5 minutes; and the solution wasdecanted. For a final time, the solids were suspended in Et₂O (20 mL);the mixture was centrifuged for 5 minutes; and the solution wasdecanted.

Cyclization Procedure:

The solids were dissolved in 20 mL MeCN:aq. 0.1M NH₄OAc (1:1), and thesolution was carefully adjusted to pH=8.5-9.0 using aq NaOH (1.0M). Thesolution was then allowed to stand (stirring not necessary) overnight(app. 18 h). 1 mL DMSO was added, and the reaction solution wasconcentrated in a SpeedVac centrifuge evaporator overnight with mildheating. Approximately 1 mL of MeOH was added to the residue, and theresulting solution was purified by the method described in theindividual examples. As an alternate cyclization procedure, the materialobtained from a 0.1 mmol scale reaction was taken up in ˜20 mL MeOHcontaining ˜5 drops of Hunig's base (pH˜10). This was left to stand atrt without stirring overnight. Solvents were removed in vacuo and theresidue purified as described in the individual examples.

General Triazole Formation Procedure for Examples 13051-13077,13120-13128, 13141-13164, and 14121-14126

To a solution (or in some cases a suspension) of the alkyne and azidecomponents in 1:1 water:tBuOH (˜0.016 M) was added 1.3 eq. (vs. peptide)of sodium(R)-2-((S)-1,2-dihydroxyethyl)-4-hydroxy-5-oxo-2,5-dihydrofuran-3-olate.Then 0.2 eq (vs. peptide) of CuSO₄ (as a 0.05 mg/μL aqueous solution)was added, and the resulting solution was stirred at rt for ˜18 h. Themixture was injected directly on a preparatory HPLC, as described in thespecific examples.

General Triazole Formation Procedure for Examples 14051-14102

The mixture of INT-1400J (48 mg, 0.023 mmol),(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate (17.64 mg, 0.028 mmol), sodium(R)-2-((S)-1,2-dihydroxyethyl)-4-hydroxy-5-oxo-2,5-dihydrofuran-3-olate(6.39 mg, 0.032 mmol) and Copper(II) sulfate pentahydrate (2.290 mg,9.17 μmol) in t-BuOH (459 μl)/Water (459 μl) was stirred at rtovernight.

Preparation of Fmoc-(S)-propargylglycine-2-chlorotrityl resin

To a solution of2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pent-4-ynoic acid (0.671 g,2.000 mmol) in 3 mL DMF and 20 mL DCM was added DIPEA (1.397 ml, 8.00mmol). The resulting solution was added to 2.0 g chlorotrityl chlorideresin (1.2 meq/g), and the resulting mixture was shaken for 2 h at rt.The solvents were filtered off, and the resin was capped with 17:2:1DCM/MeOH/DIPEA (shaken with 10 mL of the solution for 15 min thenfiltered). This was repeated twice more. The resin washed twice withDCM, 4 times with DMF, and 6 times with DCM. (each cycle was ˜10 min,followed by filtration (Buchner funnel). The resin was dried under N₂,yielding 2.2 g of resin, estimated loading of 0.9 mmol/g.

Preparation of 2-(2-(2-methoxyethoxy)ethoxy)-N-(prop-2-yn-1-yl)acetamidescheme

To a solution of 2-(2-(2-methoxyethoxy)ethoxy)acetic acid (1.53 g, 8.59mmol) in THF (28.6 ml) was added prop-2-yn-1-amine (0.660 ml, 10.30mmol) and DIPEA (3.00 ml, 17.17 mmol). HBTU (3.91 g, 10.30 mmol) wasthen added, and the mixture stirred at rt. After ˜1.5 h, LC/MS indicatedthat the reaction had advanced to near-completion. The solvent wasdecanted from the white precipitate, and was concentrated in vacuo. Theresidue was taken up in EtOAc, then extracted with NaHCO₃ to remove anyunreacted acid. The organic layer was then extracted twice with 0.1 MHCl to remove excess base. The organic extracts were then dried overMgSO₄, filtered, and concentrated in vacuo. The residue was applied tosilica gel (40 g) and eluted with CH₂Cl₂ (60 mL), then a gradient to 25%acetone/CH₂Cl₂ over 600 mL, and finally a hold at 25% acetone/CH₂Cl₂ for300 mL. The appropriate fractions were combined to obtain2-(2-(2-methoxyethoxy)ethoxy)-N-(prop-2-yn-1-yl)acetamide (102.2 mg,0.475 mmol, 5.53% yield). ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.41 (br. s.,1H), 4.11 (dd, J=5.6, 2.6 Hz, 2H), 4.05 (s, 2H), 3.74-3.67 (m, 6H),3.63-3.60 (m, 2H), 3.43 (s, 3H), 2.23 (t, J=2.5 Hz, 1H).

Preparation of N-(prop-2-yn-1-yl)stearamide scheme

To a solution of tetradecanoyl chloride (200 mg, 0.810 mmol) inTetrahydrofuran (2026 μl) was added prop-2-yn-1-amine (208 μl, 3.24mmol). After the weekend, the desired product was found by LC/MS. Excesssolvent was removed in vacuo, and water was added. The pH was adjustedto ˜10 with 1M NaOH, and the mixture extracted 3 times into CH₂Cl₂. Thecombined organic extracts were dried over MgSO4, filtered, andconcentrated in vacuo. Chromatography with a EtOAc/Hexane gradientyielded the desired material. ¹H NMR (500 MHz, CHLOROFORM-d) δ 5.57 (br.s., 1H), 4.08 (dd, J=5.3, 2.5 Hz, 2H), 2.25 (t, J=2.6 Hz, 1H), 2.23-2.19(m, 2H), 1.65 (m, 6H), 1.31 (m, 24H), 0.92-0.87 (m, 3H).

Preparation of N-(5-azidopentyl)-2-(2-(2-methoxyethoxy)ethoxy)acetamidescheme

To a solution of 2-(2-(2-methoxyethoxy)ethoxy)acetic acid (400 mg, 2.245mmol) in THF (7483 μl) was added 5-azidopentan-1-amine (317 mg, 2.469mmol) and DIPEA (784 μl, 4.49 mmol). HBTU (936 mg, 2.469 mmol) was thenadded, and the mixture stirred at rt. After ˜1.5 h, LC/MS indicated thatthe reaction had advanced to near-completion. The solvent was decantedfrom the white precipitate, and was concentrated in vacuo. The residuewas taken up in EtOAc, then extracted with NaHCO₃ to remove anyunreacted acid. The organic layer was then extracted twice with 0.1 MHCl to remove excess base. The organic extracts were then dried overMgSO4, filtered, and concentrated in vacuo. The material was used as-isfor further chemistry. LC/MS: (M+H)⁺=289.15.

(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate

Step 1: Preparation of(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate

The mixture of 5-azidopentan-1-amine (0.320 g, 2.419 mmol), Vitamin Esuccinate (1.07 g, 2.016 mmol), DIPEA (0.704 ml, 4.03 mmol) and HBTU(0.765 g, 2.016 mmol) in THF (6.72 ml) was stirred at rt overnight. Theresulting crude product was purified by Biotage (silic gel, 300 g, 0 to20% acetone/CH₂Cl₂) to get(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate (1.29 g, 2.013 mmol, 100%yield). Analysis condition D: Retention time=4.87 min; ESI-MS(+) m/z641.4 (M+H)⁺; ¹H NMR (500 MHz, METHANOL-d₄) δ 3.26-3.15 (m, 4H), 2.95(t, J=6.7 Hz, 2H), 2.62 (dt, J=12.8, 6.6 Hz, 4H), 2.13-2.06 (m, 3H),1.99-1.96 (m, 3H), 1.84-1.81 (m, 3H), 1.90-1.76 (m, 2H), 1.69-1.02 (m,30H), 0.98-0.79 (m, 12H)

2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate

Step 1: Preparation of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate

The mixture of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanol int-butylmethyl ether (3.77 ml, 1.884 mmol), Vitamin E succinate (1.0 g,1.884 mmol), DMAP (0.092 g, 0.754 mmol) and EDCI (1.138 g, 5.93 mmol) inCH₂Cl₂ (11.35 ml) was stirred at rt overnight. The resulting crudeproduct was purified by Biotage (silic gel, 300 g, 0 to 20%acetone/CH₂Cl₂) to get 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate (1.37 g, 1.872 mmol, 99% yield). Analysis condition D:Retention time=5.18 min; ESI-MS(+) m/z 732.5 (M+H)⁺

¹H NMR (500 MHz, METHANOL-d₄) δ 4.31-4.21 (m, 2H), 3.74-3.60 (m, 12H),3.38-3.34 (m, 2H), 2.95 (dd, J=7.5, 5.3 Hz, 2H), 2.78 (dd, J=7.4, 5.4Hz, 2H), 2.64 (t, J=6.8 Hz, 2H), 2.12-2.07 (m, 3H), 2.01 (s, 3H),1.99-1.94 (m, 3H), 1.90-1.75 (m, 2H), 1.66-1.03 (m, 24H), 0.94-0.83 (m,12H)

17-azido-3,6,9,12,15-pentaoxaheptadecyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate

Step 1: Preparation of 17-hydroxy-3,6,9,12,15-pentaoxaheptadecyl4-methylbenzenesulfonate

3,6,9,12,15-Pentaoxaheptadecane-1,17-diol (8 g, 28.3 mmol) was dissolvedin THF (30 mL). pyridine (7.13 mL, 88 mmol) was added to the mixturefollowed by 4-methylbenzene-1-sulfonyl chloride (5.41 g, 28.4 mmol).Mixture was stirred at room temperature for 3 hours. Mixture wasconcentrated by roto-vap. The resulting residue was dissolved indichloromethane, washed twice with saturated aqueous sodium bicarbonate.The combined aqueous layers were back-extracted with dichloromethane.The combined organics were washed twice with 1N hydrochloric acid andonce with brine. Organics were dried over MgSO₄, filtered, concentratedto dryness to get 17-hydroxy-3,6,9,12,15-pentaoxaheptadecyl4-methylbenzenesulfonate (5.10 g, 11.68 mmol, 41.2% yield) which wasused as is in the next step. Analysis condition D: Retention time=1.37min; ESI-MS(+) m/z 437.3 (M+H)⁺

Step 2: Preparation of 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol

17-Hydroxy-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate (5.10g, 11.68 mmol) was dissolved in EtOH (37.4 ml). Sodium azide (2.97 g,45.7 mmol) was added to the mixture followed by water (1.498 ml).Mixture was warmed to reflux and held with stirring for 15 hours. Thecloudy reaction mixture was concentrated by roto-vap. The residue wastreated with water. The mixture was extracted twice withdichloromethane. The combined organics were washed twice with aqueoussodium bicarbonate. Organics were dried over MgSO₄, filtered and thenconcentrated to dryness. The residue was purified via Biotage (Silica;300 g; 0 to 9% D MeOH/Dichloromethane over 2400 mL). All effluent wascollected in 16×150 culture tubes. Major peak fractions, as determinedby TLC (silica; 5% MeOH—CH₂Cl₂; iodine chamber) was isolated andconcentrated to dryness. 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol(3.08 g, 10.02 mmol, 86% yield) was obtained as clear colorless oil.Analysis condition D: Retention time=1.19 min; ESI-MS(+) m/z 330.2(M+Na); ¹H NMR (500 MHz, METHANOL-d₄) δ 3.73-3.61 (m, 20H), 3.60-3.55(m, 2H), 3.39 (t, J=4.9 Hz, 2H).

Step 3: Preparation of 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol

The mixture of 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol (0.579 g,1.884 mmol), Vitamin E succinate (1.0 g, 1.884 mmol), DMAP (0.092 g,0.754 mmol) and EDCI (1.138 g, 5.93 mmol) in CH₂Cl₂ (11.35 ml) wasstirred at rt overnight. The resulting crude product was purified byBiotage (SG, 300 g, 0 to 40% acetone/CH₂Cl₂) to get17-azido-3,6,9,12,15-pentaoxaheptadecyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate (1.20 g, 1.463 mmol, 78% yield). Analysis condition D:Retention time=5.50 min; ESI-MS(+) m/z 842.6 (M+Na).

¹H NMR (500 MHz, METHANOL-d₄) δ 4.31-4.23 (m, 2H), 3.73-3.70 (m, 2H),3.69-3.59 (m, 18H), 3.40-3.30 (m, 2H), 2.98-2.91 (m, 2H), 2.80-2.75 (m,2H), 2.64 (t, J=6.8 Hz, 2H), 2.09 (s, 3H), 2.01 (s, 3H), 1.98 (s, 3H),1.89-1.76 (m, 2H), 1.61-1.50 (m, 4H), 1.48-1.01 (m, 20H), 0.94-0.83 (m,12H).

23-azido-3,6,9,12,15,18,21-heptaoxatricosyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate

Step 1: Preparation of 23-hydroxy-3,6,9,12,15,18,21-heptaoxatricosyl4-methylbenzenesulfonate

3,6,9,12,15,18,21-Heptaoxatricosane-1,23-diol (5.5 g, 14.85 mmol) wasdissolved in THF (30 mL). pyridine (3.73 mL, 46.2 mmol) was added to themixture followed by 4-methylbenzene-1-sulfonyl chloride (2.84 g, 14.88mmol). The mixture stirred at room temperature overnight. Mixture wasconcentrated by roto-vap. Residue was dissolved in dichloromethane,washed twice with saturated aqueous sodium bicarbonate. The combinedaqueous layers were back-extracted with dichloromethane. The combinedorganics were washed twice with 1N hydrochloric acid and once withbrine. The organics were dried MgSO₄, filtered, concentrated to drynessto get 23-hydroxy-3,6,9,12,15,18,21-heptaoxatricosyl4-methylbenzenesulfonate (2.58 g, 4.92 mmol, 33.1% yield) which is usedas is in the next step. Analysis condition D: Retention time=1.41 min;ESI-MS(+) m/z 525.3 (M+H)⁺

Step 2: Preparation of 23-azido-3,6,9,12,15,18,21-heptaoxatricosan-1-ol

23-Hydroxy-3,6,9,12,15,18,21-heptaoxatricosyl 4-methylbenzenesulfonate(2.58 g, 4.92 mmol) was dissolved in EtOH (15.76 ml). Sodium azide(1.250 g, 19.23 mmol) was added to the mixture followed by water (0.630ml). Mixture was warmed to reflux and held with stirring for 15 hours.The cloudy reaction mixture was concentrated by roto-vap. Residue wastreated with water. Material was extracted twice with dichloromethane.Combined organics were washed twice with aqueous sodium bicarbonate.Organics were dried MgSO₄, filtered and then concentrated to dryness.The residue was purified via Biotage (Silica; 300 g; 0 to 10%MeOH/Dichloromethane over 2400 mL). All effluent was collected in 16×150culture tubes. Major peak fractions, as determined by TLC (silica; 5%MeOH—CH₂Cl₂; iodine chamber) was isolated and concentrated to dryness.23-azido-3,6,9,12,15,18,21-heptaoxatricosan-1-ol (1.55 g, 3.92 mmol, 80%yield) was obtained as clear colorless oil. Analysis condition D:Retention time=1.18 min; ESI-MS(+) m/z 396.3 (M+H)⁺; ¹H NMR (500 MHz,METHANOL-d₄) δ 3.75-3.60 (m, 28H), 3.60-3.54 (m, 2H), 3.43-3.37 (m, 2H).

Step 3: Preparation of 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol

The mixture of 23-azido-3,6,9,12,15,18,21-heptaoxatricosan-1-ol (0.745g, 1.884 mmol), Vitamin E succinate (1.0 g, 1.884 mmol), DMAP (0.092 g,0.754 mmol) and EDCI (1.138 g, 5.93 mmol) in CH₂Cl₂ (11.35 ml) wasstirred at rt overnight. The resulting crude product was purified byBiotage (SG, 300 g, 0 to 50% acetone/CH₂Cl₂) to get23-azido-3,6,9,12,15,18,21-heptaoxatricosyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate (0.77 g, 0.848 mmol, 45.0% yield).

Analysis condition D: Retention time=5.04 min; ESI-MS(+) m/z 908.9(M+H)⁺¹H NMR (500 MHz, METHANOL-d₄) δ 4.30-4.23 (m, 2H), 3.77-3.56 (m,28H), 3.42-3.35 (m, 2H), 2.96 (dd, J=7.5, 5.3 Hz, 2H), 2.84-2.73 (m,2H), 2.64 (t, J=6.8 Hz, 2H), 2.09 (s, 3H), 2.00 (d, J=17.1 Hz, 6H), 1.83(dq, J=18.4, 6.7 Hz, 2H), 1.64-1.04 (m, 24H), 0.97-0.79 (m, 12H).

23-azido-3,6,9,12,15,18,21-heptaoxatricosyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate

Step 1: Preparation of 2,5,8,11-tetraoxatridecan-13-yl4-methylbenzenesulfonate

2,5,8,11-Tetraoxatridecan-13-ol (5.0 g, 24.01 mmol) was dissolved in THF(20.01 ml). pyridine (5.83 ml, 72.0 mmol) was added to the mixturefollowed by 4-methylbenzene-1-sulfonyl chloride (5.49 g, 28.8 mmol). Themixture stirred at room temperature overnight. Mixture was concentratedby roto-vap. Residue was dissolved in dichloromethane. Material washedtwice with saturated aqueous sodium bicarbonate. Combined aqueous layerswere back-extracted with dichloromethane. Combined organics were washedtwice with 1N hydrochloric acid and once with brine. Organics were driedMgSO₄, filtered and then concentrated to dryness to get2,5,8,11-tetraoxatridecan-13-yl 4-methylbenzenesulfonate (4.12 g, 11.37mmol, 47.3% yield) which was used as is in the next step.

Step 2: Preparation of 13-azido-2,5,8,11-tetraoxatridecane

2,5,8,11-Tetraoxatridecan-13-yl 4-methylbenzenesulfonate (4.12 g, 11.37mmol) was dissolved in EtOH (18.22 ml). Sodium azide (1.478 g, 22.73mmol) was added to the mixture followed by water (0.729 ml). The mixturewas warmed to reflux and held with stirring for 15 hours. The cloudyreaction mixture was concentrated by roto-vap. Residue was treated withwater. Material was extracted twice with dichloromethane. Combinedorganics were washed twice with aqueous sodium bicarbonate. Organicswere dried MgSO₄, filtered and then concentrated to dryness. The residuewas purified via Biotage (Silica; 300 g; 0 to 9% D MeOH/Dichloromethaneover 2400 mL). All effluent was collected in 16×150 culture tubes. Majorpeak fractions, as determined by TLC (silica; 5% MeOH—CH₂Cl₂; iodinechamber) was isolated and concentrated to dryness.13-azido-2,5,8,11-tetraoxatridecane (1.17 g, 5.02 mmol, 44.1% yield) wasobtained as clear colorless oil. [M+H]⁺ at m/z 234, and sodium adduct[M+Na]⁺ at m/z 256; ¹H NMR (400 MHz, METHANOL-d4) δ 3.74-3.60 (m, 12H),3.58-3.53 (m, 2H), 3.43-3.35 (m, 5H).

(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid

Step 1: Preparation of 16-(tert-butoxy)-16-oxohexadecanoic acid

Hexadecanedioic acid (4.5 g, 15.71 mmol) was suspended in Toluene (28.1ml) and the mixture was heated to reflux.1,1-di-tert-butoxy-N,N-dimethylmethanamine (10.10 ml, 42.1 mmol) wasadded drop-wise over 30 min. The mixture was reflux overnight. Thesolvent was removed in vacuo at 50° C. and the crude material wassuspended in CH₂Cl₂/EtOAc (75 mL. 1:1) and stirred for 15 min. Thesolids were removed by filtration and washed with CH₂Cl₂ (25 mL). Thefiltration was evaporated in vacuo. The resulting material was suspendedin CH₂Cl₂ (6 mL), cooled with ice for 10 mins, and filtered. The solventwas removed in vacuo to leave crude product which was purified by flashchromatography (silic gel, EtOAc/Hexane) to get16-(tert-butoxy)-16-oxohexadecanoic acid (2.56 g, 7.47 mmol, 47.6%yield). Analysis condition D: Retention time=5.04 min; ESI-MS(+) m/z269.3 [M−OC(CH₃)_(3];) ¹H NMR (500 MHz, METHANOL-d₄) δ 2.33-2.18 (m,4H), 1.66-1.54 (m, 4H), 1.50-1.43 (m, 9H), 1.40-1.25 (m, 20H).

Step 2: Preparation of (S)-methyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-azidobutanoate

To a mixture of (2S)—N-FMOC-4-AZIDO-BUTANOIC ACID (1.0 g, 2.73 mmol) inMeOH (4.21 ml)/CH₂Cl₂ (12.64 ml) was added (TRIMETHYLSILYL)DIAZOMETHANEin diethyl ether (2.047 ml, 4.09 mmol). The resulting mixture wasstirred at rt for 2 h. The mixture was concentrated to get (S)-methyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-azidobutanoate, whichwas used as is in the next step. ¹H NMR (500 MHz, METHANOL-d₄) δ 7.81(d, J=7.6 Hz, 2H), 7.73-7.63 (m, 2H), 7.47-7.22 (m, 4H), 4.41 (d, J=6.7Hz, 2H), 4.35-4.17 (m, 2H), 3.82-3.70 (m, 3H), 3.49-3.34 (m, 2H),2.21-2.04 (m, 1H), 1.97-1.80 (m, 1H).

Step 3: Preparation of (S)-methyl4-azido-2-((tert-butoxycarbonyl)amino)butanoate

The mixture of (S)-methyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-azidobutanoate (1.038 g,2.73 mmol) and diethylamine (4.0 mL, 38.3 mmol) in CH₂Cl₂ (4 mL) wasstirred at rt for 2 h. LCMS showed disappearance of S.M. and productformation along with FMOC related peaks. Concentrated and the resultingproduct was used as is in the next step. ¹H NMR (500 MHz, METHANOL-d4)83.85-3.69 (m, 3H), 3.63-3.52 (m, 1H), 3.51-3.40 (m, 2H), 2.05-1.91 (m,1H), 1.88-1.76 (m, 1H).

Step 4: Preparation of (S)-tert-butyl16-((4-azido-1-methoxy-1-oxobutan-2-yl)amino)-16-oxohexadecanoate

The mixture of (S)-methyl 2-amino-4-azidobutanoate (0.432 g, 2.73 mmol),16-(tert-butoxy)-16-oxohexadecanoic acid (0.935 g, 2.73 mmol), DIPEA(1.907 ml, 10.92 mmol) and HBTU (1.035 g, 2.73 mmol) in THF (27.3 ml)was stirred at rt overnight. The resulting crude product was purified byBiotage (SG, 300 g, 0 to 10% acetone/CH₂Cl₂) to get (S)-tert-butyl16-((4-azido-1-methoxy-1-oxobutan-2-yl)amino)-16-oxohexadecanoate (1.3g, 2.69 mmol, 99% yield). Analysis condition D: Retention time=2.62 min;ESI-MS(+) m/z 483.3 (M+H)⁺

Step 5: Preparation of(S)-4-azido-2-(16-(tert-butoxy)-16-oxohexadecanamido)butanoic acid

(S)-tert-Butyl16-((4-azido-1-methoxy-1-oxobutan-2-yl)amino)-16-oxohexadecanoate (1.3g, 2.69 mmol), was dissolved in THF (13.47 ml) followed by the additionof LiOH (0.323 g, 13.47 mmol) and Water (13.47 ml). The reaction wasstirred at rt for 3 h. Concentrated the reaction mixture to dryness. Theresulting (S)-4-azido-2-(16-(tert-butoxy)-16-oxohexadecanamido)butanoicacid was used as is in the next step.

Analysis condition D: Retention time=2.56 min; ESI-MS(+) m/z 469.4(M+H)⁺

Step 6: Preparation of(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid

The mixture of(S)-4-azido-2-(16-(tert-butoxy)-16-oxohexadecanamido)butanoic acid (1261mg, 2.69 mmol) and TFA (3 mL, 38.9 mmol) in DCM (20 mL) was stirred atrt for 2 h. The resulting crude product was purified by Prep-HPLC(Solvent A=10% MeOH—90% H₂O—0.1% TFA, Solvent B=90% MeOH—10% H2O—0.1%TFA. Column: PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40 ml/min,50-100% B, 10 min and stop at 12 min) to obtain(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (326 mg,0.790 mmol, 29.4% yield). Analysis condition D: Retention time=2.30 min;ESI-MS(+) m/z 413.3 (M+H)⁺; ¹H NMR (500 MHz, METHANOL-d₄) δ 4.52 (dd,4.7 Hz, 1H), 3.53-3.35 (m, 2H), 2.34-2.21 (m, 4H), 2.19-2.07 (m, 1H),1.99-1.84 (m, 1H), 1.63 (dquin, J=14.1, 7.1 Hz, 4H), 1.48-1.17 (m, 20H).

(S)-methyl 4-azido-2-palmitamidobutanoate

Step 1: Preparation of (S)-methyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-azidobutanoate

To a mixture of (2S)—N-FMOC-4-AZIDO-BUTANOIC ACID (1.0 g, 2.73 mmol) inMeOH (4.21 ml)/CH₂Cl₂ (12.64 ml) was added (TRIMETHYLSILYL)DIAZOMETHANEin diethyl ether (2.047 ml, 4.09 mmol). The resulting mixture wasstirred at rt for 2 h. The mixture was concentrated to get (S)-methyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-azidobutanoate, whichwas used as is in the next step. ¹H NMR (500 MHz, METHANOL-d₄) δ 7.81(d, J=7.6 Hz, 2H), 7.73-7.63 (m, 2H), 7.47-7.22 (m, 4H), 4.41 (d, J=6.7Hz, 2H), 4.35-4.17 (m, 2H), 3.82-3.70 (m, 3H), 3.49-3.34 (m, 2H),2.21-2.04 (m, 1H), 1.97-1.80 (m, 1H).

Step 2: Preparation of (S)-methyl4-azido-2-((tert-butoxycarbonyl)amino)butanoate

The mixture of (S)-methyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-azidobutanoate (1.038 g,2.73 mmol) and diethylamine (4.0 mL, 38.3 mmol) in CH₂Cl₂ (4 mL) wasstirred at rt for 2 h. LCMS showed disappearance of S.M. and productformation along with FMOC related peaks. Concentrated and the resultingproduct was used as is in the next step. ¹H NMR (500 MHz, METHANOL-d₄) δ3.85-3.69 (m, 3H), 3.63-3.52 (m, 1H), 3.51-3.40 (m, 2H), 2.05-1.91 (m,1H), 1.88-1.76 (m, 1H).

Step 3: Preparation of (S)-methyl 4-azido-2-palmitamidobutanoate

The mixture of (S)-methyl 2-amino-4-azidobutanoate, TFA (544 mg, 2.00mmol), palmitic acid (513 mg, 2.000 mmol), DIPEA (1397 μl, 8.00 mmol)and HBTU (758 mg, 2.000 mmol) in THF (6667 μl) was stirred at rtovernight. The resulting crude product was purified by Biotage (SG, 300g, 0 to 85% EtOAc/Hexane) to get (S)-methyl4-azido-2-palmitamidobutanoate (477 mg, 1.203 mmol, 60.1% yield).Analysis condition D: Retention time=2.75 min; ESI-MS(+) m/z 397.3(M+H)⁺; ¹H NMR (500 MHz, METHANOL-d₄) δ 4.53 (dd, J=9.2, 5.0 Hz, 1H),3.74 (s, 3H), 3.51-3.35 (m, 2H), 2.31-2.22 (m, 2H), 2.15-2.06 (m, 1H),1.97-1.86 (m, 1H), 1.70-1.57 (m, 2H), 1.41-1.28 (m, 24H), 0.94-0.84 (m,3H).

Step 4: Preparation of (S)-methyl 4-azido-2-palmitamidobutanoate

(S)-Methyl 4-azido-2-palmitamidobutanoate (477 mg, 1.203 mmol), wasdissolved in THF (6014 μl) followed by the addition of LiOH (144 mg,6.01 mmol) and water (6014 μl). The reaction was stirred at rt for 3 h.Concentrated the reaction mixture to dryness. Diluted the residue withwater and added 1 N HCl to acidified. Extracted with CH₂Cl₂ (×3). Theorganic layer was collected, dried over MgSO4, filtered and concentratedto get (S)-4-azido-2-palmitamidobutanoic acid (440 mg, 1.150 mmol, 96%yield). Analysis condition D: Retention time=2.71 min; ESI-MS(+) m/z383.3 (M+H)⁺

¹H NMR (500 MHz, METHANOL-d4) δ 4.56-4.47 (m, 1H), 3.52-3.34 (m, 2H),2.32-2.22 (m, 2H), 2.14 (dddd, J=14.3, 7.8, 6.9, 4.9 Hz, 1H), 2.01-1.86(m, 1H), 1.72-1.56 (m, 2H), 1.49-1.12 (m, 24H), 1.01-0.80 (m, 3H).

(S)-1-azido-40-carboxy-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicAcid

Step 1: Preparation of 18-(tert-butoxy)-18-oxooctadecanoic acid

Octadecanoic acid (7.5 g, 23.85 mmol) was suspended in toluene (42.6 ml)and the mixture was heated to reflux.1,1-di-tert-butoxy-N,N-dimethylmethanamine (15.33 ml, 63.9 mmol) wasadded drop-wise over 30 min. The mixture was reflux overnight. Thesolvent was removed in vacuo at 50° C. and the crude material wassuspended in CH₂Cl₂/EtOAc (110 mL. 1:1) and stirred for 15 min. Thesolids were removed by filtration and washed with CH₂Cl₂ (40 mL). Thefiltration was evaporated in vacuo. The crude product was purified byflash chromatography (SG, 0 to 25% Acetone/CH₂Cl₂) to get18-(tert-butoxy)-18-oxooctadecanoic acid (3.95 g, 10.66 mmol, 44.7%yield).

Analysis condition D: Retention time=5.04 min; ESI-MS(+) m/z 297.3[M−OC(CH₃)_(3])

¹H NMR (500 MHz, METHANOL-d₄) δ 2.29 (t, J=7.5 Hz, 2H), 2.22 (t, J=7.4Hz, 2H), 1.67-1.53 (m, 4H), 1.50-1.42 (m, 9H), 1.40-1.25 (m, 24H).

Step 2: Preparation of 1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl)octadecanedioate

DCC (5.11 ml, 5.11 mmol) was added to a solution of18-(tert-butoxy)-18-oxooctadecanoic acid (1.72 g, 4.64 mmol) and1-hydroxypyrrolidine-2,5-dione (0.588 g, 5.11 mmol) in DMF (48 mL). Themixture was stirred at rt overnight. The mixture was filtered andconcentrated to get 1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl)octadecanedioate which was used as is in the next step.

Step 3: Preparation of(S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid

Water (5.80 ml) was added to a mixture of(S)-4-amino-5-(tert-butoxy)-5-oxopentanoic acid (1.038 g, 5.11 mmol),1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl) octadecanedioate (2.171 g,4.64 mmol), SODIUM BICARBONATE (0.468 g, 5.57 mmol) in THF (17.41 ml).The resulting clear solution was stirred at rt for 4 h. All THF wasremoved, HCl (6.04 ml, 6.04 mmol) was added and the pH was adjusted to2-3 at 0° C. The resulting suspension was extracted with CH₂Cl₂ (×3),The organic layer was concentrated. The resulting crude product waspurified by flash chromatography (acetone/CH₂Cl₂ 0 to 25%) to afford(S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (2.29 g, 4.12 mmol, 89% yield) as a white solid. Analysis conditionD: Retention time=2.74 min; ESI-MS(+) m/z 555.6 (M+H)⁺

¹H NMR (500 MHz, METHANOL-d₄) δ 4.32 (dd, J=9.0, 5.3 Hz, 1H), 2.45-2.33(m, 2H), 2.30-2.06 (m, 5H), 1.99-1.82 (m, 3H), 1.78-1.53 (m, 2H),1.53-1.44 (m, 18H), 1.44-1.26 (m, 24H).

Step 4: Preparation of (S)-tert-butyl1-azido-40-(tert-butoxycarbonyl)-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oate

To a solution of(S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (438 mg, 0.789 mmol) in DMF (1593 μl) was added Hunig's Base (275μl, 1.577 mmol) and HATU (400 mg, 1.051 mmol).35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontan-1-amine(300 mg, 0.526 mmol) was then added, and the solution stirred at rt. Themixture was stirred overnight.

The mixture was poured into water and extracted 3 times into CH₂Cl₂. Thecombined organic extracts were dried over MgSO4, filtered, andconcentrated in vacuo to get (S)-tert-butyl1-azido-40-(tert-butoxycarbonyl)-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oatewhich is used as is in the next step. Analysis condition D: Retentiontime=2.84 min; ESI-MS(+) m/z 1109.1 (M+H)⁺

Step 5: Preparation of(S)-1-azido-40-carboxy-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicAcid

The mixture of (S)-tert-butyl1-azido-40-(tert-butoxycarbonyl)-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oate(280 mg, 0.253 mmol) and TFA (3 mL, 38.9 mmol) in DCM (3.0 mL) wasstirred at rt for 2 h. The resulting crude product was purified byPrep-HPLC (Solvent A=10% MeOH—90% H2O—0.1% TFA, Solvent B=90% MeOH—10%H₂O—0.1% TFA. Column: PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40ml/min, 50-100% B, 10 min and stop at 12 min) to obtain(S)-1-azido-40-carboxy-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicacid (124 mg, 0.124 mmol, 49.3% yield). Analysis condition D: Retentiontime=2.43 min; ESI-MS(+) m/z 996.9 (M+H)⁺; ¹H NMR (500 MHz, METHANOL-d4)δ 4.44-4.35 (m, 1H), 3.84-3.27 (m, 48H), 2.39-2.11 (m, 7H), 2.04-1.87(m, 1H), 1.71-1.55 (m, 4H), 1.44-1.18 (m, 24H).

(S)-1-azido-16-carboxy-13,18-dioxo-3,6,9-trioxa-12,17-diazapentatriacontan-35-oicacid

Step 1: To a solution of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanamine(113 mg, 0.517 mmol) in DMF (4498 μl) was added Hunig's Base (314 μl,1.799 mmol), then(S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (250 mg, 0.450 mmol). HATU (342 mg, 0.900 mmol) was then added, andthe resulting solution was stirred at rt. LC/MS showed conversion to thedesired m/z. Removed DMF on high vacuum, then the residue was applied tosilica gel (40 g) and eluted with DCM (100 mL), then a gradient to 75%DCM/acetone over 540 mL and finally a hold at 75% DCM/acetone for 150mL. The desired fractions were combined. The material was taken into thenext step as is.Step 2: To a solution of (S)-tert-butyl1-azido-16-(tert-butoxycarbonyl)-13,18-dioxo-3,6,9-trioxa-12,17-diazapentatriacontan-35-oate(414.0 mg, 0.548 mmol) in DCM (5476 μl) was added TFA (1266 μl, 16.43mmol). LC/MS indicated a slow reaction, so another 14 eq TFA was addedand the mixture stirred further. After another ˜6 h, LC/MS indicated anearly complete reaction. Solvents were removed in vacuo. The mixturewas taken up in Hunig's base/MeOH (˜1%). The reaction mixture waspurified by PREP HPLC in 5 injections: (30×100 mm HPLC Luna Axia C18 50to 100% A:B over 10 min, 5 min at 100% B (A is 90:10:0.1 water:MeOH:TFA;B is 90:10:0.1 MeOH:water:TFA)). The desired fractions were combined andconcentrated to afford(S)-1-azido-16-carboxy-13,18-dioxo-3,6,9-trioxa-12,17-diazapentatriacontan-35-oicacid (112.4 mg, 0.124 mmol, 22.64% yield). LC/MS: (M+H)⁺=644.45.

(S)-1-azido-22-carboxy-19,24-dioxo-3,6,9,12,15-pentaoxa-18,23-diazahentetracontan-41-oicAcid

Step 1: Preparation of 18-(tert-butoxy)-18-oxooctadecanoic acid

OCTADECANEDIOIC ACID (7.5 g, 23.85 mmol) was suspended in toluene (42.6ml) and the mixture was heated to reflux.1,1-di-tert-butoxy-N,N-dimethylmethanamine (15.33 ml, 63.9 mmol) wasadded dropwise over 30 min. The mixture was reflux overnight. Thesolvent was removed in vacuo at 50° C. and the crude material wassuspended in CH₂Cl₂/EtOAc (110 mL. 1:1) and stirred for 15 min. Thesolids were removed by filtration and washed with CH₂Cl₂ (40 mL). Thefiltration was evaporated in vacuo. The crude product was purified byflash chromatography (SG, 0 to 25% acetone/CH₂Cl₂) to get18-(tert-butoxy)-18-oxooctadecanoic acid (3.95 g, 10.66 mmol, 44.7%yield). Analysis condition D: Retention time=5.04 min; ESI-MS(+) m/z297.3 [M−OC(CH₃)_(3];) ¹H NMR (500 MHz, METHANOL-d₄) δ 2.29 (t, J=7.5Hz, 2H), 2.22 (t, J=7.4 Hz, 2H), 1.67-1.53 (m, 4H), 1.50-1.42 (m, 9H),1.40-1.25 (m, 24H).

Step 2: Preparation of 1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl)octadecanedioate

DCC (5.11 ml, 5.11 mmol) was added to a solution of18-(tert-butoxy)-18-oxooctadecanoic acid (1.72 g, 4.64 mmol) and1-hydroxypyrrolidine-2,5-dione (0.588 g, 5.11 mmol) in DMF (48 mL). Themixture was stirred at rt overnight. The mixture was filtered andconcentrated to get 1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl)octadecanedioate which was used as is in the next step.

Step 3: Preparation of(S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid

Water (5.80 ml) was added to a mixture of(S)-4-amino-5-(tert-butoxy)-5-oxopentanoic acid (1.038 g, 5.11 mmol),1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl) octadecanedioate (2.171 g,4.64 mmol), SODIUM BICARBONATE (0.468 g, 5.57 mmol) in THF (17.41 ml).The resulting clear solution was stirred at rt for 4 h. All THF wasremoved, HCl (6.04 ml, 6.04 mmol) was added and the pH was adjusted to2-3 at 0° C. The resulting suspension was extracted with CH₂Cl₂ (×3).The organic layer was concentrated. The resulting crude product waspurified by flash chromatography (acetone/CH₂Cl₂ 0 to 25%) to afford(S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (2.29 g, 4.12 mmol, 89% yield) as a white solid. Analysis conditionD: Retention time=2.74 min; ESI-MS(+) m/z 555.6 (M+H)⁺¹H NMR (500 MHz,METHANOL-d₄) δ 4.32 (dd, J=9.0, 5.3 Hz, 1H), 2.45-2.33 (m, 2H),2.30-2.06 (m, 5H), 1.99-1.82 (m, 3H), 1.78-1.53 (m, 2H), 1.53-1.44 (m,18H), 1.44-1.26 (m, 24H).

Step 4: Preparation of (S)-tert-butyl1-azido-22-(tert-butoxycarbonyl)-19,24-dioxo-3,6,9,12,15-pentaoxa-18,23-diazahentetracontan-41-oate

To a solution of(S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (225 mg, 0.405 mmol) in DMF (4048 μl) was added Hunig's Base (212μl, 1.214 mmol) and HATU (308 mg, 0.810 mmol).17-azido-3,6,9,12,15-pentaoxaheptadecan-1-amine, HCl (139 mg, 0.405mmol) was then added, and the solution stirred at rt. The crude productwas purified by flash chromatography (220 g, silic gel, 10 to 60%Acetone/CH₂Cl₂) to get (S)-tert-butyl1-azido-22-(tert-butoxycarbonyl)-19,24-dioxo-3,6,9,12,15-pentaoxa-18,23-diazahentetracontan-41-oate(330 mg, 0.391 mmol, 97% yield). Analysis condition D: Retentiontime=2.88 min; ESI-MS(+) m/z 844.7 (M+H)⁺; ¹H NMR (500 MHz, METHANOL-d₄)δ 4.30-4.23 (m, 1H), 3.74-3.60 (m, 18H), 3.60-3.52 (m, 2H), 3.43-3.35(m, 4H), 2.34-2.28 (m, 2H), 2.28-2.19 (m, 4H), 2.15-2.08 (m, 1H),1.98-1.87 (m, 1H), 1.69-1.53 (m, 4H), 1.52-1.44 (m, 18H), 1.41-1.27 (m,24H).

Step 5: Preparation(S)-1-azido-22-carboxy-19,24-dioxo-3,6,9,12,15-pentaoxa-18,23-diazahentetracontan-41-oicAcid

The mixture of (S)-tert-butyl1-azido-22-(tert-butoxycarbonyl)-19,24-dioxo-3,6,9,12,15-pentaoxa-18,23-diazahentetracontan-41-oate(330 mg, 0.391 mmol) and TFA (0.422 mL, 5.47 mmol) in DCM (3.0 mL) wasstirred at rt for 2 h. The resulting crude product was purified byPrep-HPLC (Solvent A=10% MeOH—90% H2O—0.1% TFA, Solvent B=90% MeOH—10%H₂O—0.1% TFA. Column: PHENOMENEX LUNA 30×150 mm, S10, Flow rate: 40ml/min, 50-100% B, 10 min and stop at 13 min) to obtain(S)-1-azido-22-carboxy-19,24-dioxo-3,6,9,12,15-pentaoxa-18,23-diazahentetracontan-41-oicacid (101 mg, 0.138 mmol, 35.3% yield). Analysis condition D: Retentiontime=2.42 min; ESI-MS(+) m/z 732.5 (M+H)⁺

(S)-1-azido-28-carboxy-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oicAcid

Step 1 To a solution of23-azido-3,6,9,12,15,18,21-heptaoxatricosan-1-amine (204 mg, 0.517 mmol)in DMF (4498 μl) was added Hunig's Base (314 μl, 1.799 mmol), then(S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (250 mg, 0.450 mmol). HATU (342 mg, 0.900 mmol) was then added, andthe resulting solution was stirred at rt. LC/MS showed conversion to thedesired m/z. Removed DMF on high vacuum, then the residue was applied tosilica gel (40 g) and eluted with DCM (90 mL), then a gradient to 75%DCM/acetone over 540 mL and finally a hold at 75% DCM/acetone for 150mL. The desired fractions were combined to obtain (S)-tert-butyl1-azido-28-(tert-butoxycarbonyl)-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oate(394.2 mg, 0.423 mmol, 94% yield).Step 2: To a solution of(S)-tert-butyl1-azido-28-(tert-butoxycarbonyl)-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oate(394.2 mg, 0.423 mmol) in DCM (4229 μl) was added TFA (456 μl, 5.92mmol). LC/MS indicated a slow reaction, so another 14 eq TFA was addedand the mixture stirred further. After another ˜6 h, LC/MS indicated anearly complete reaction. Solvents were removed in vacuo. The mixturewas taken up in MeOH. The reaction mixture was purified by PREP HPLC in7 injections: (30×100 mm HPLC Luna Axia C18 50 to 100% A:B over 10 min,5 min at 100% B (A is 90:10:0.1 water:MeOH:TFA; B is 90:10:0.1MeOH:water:TFA)). Speedvac'd appropriate fractions. Isolated(S)-1-azido-28-carboxy-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oicacid (121.0 mg, 0.148 mmol, 34.9% yield). LC/MS: (M+H)⁺=820.60. ¹H NMR(500 MHz, CHLOROFORM-d) δ 7.30 (d, J=6.4 Hz, 1H), 7.04 (t, J=5.2 Hz,1H), 4.51 (q, J=6.2 Hz, 1H), 3.72-3.64 (m, 27H), 3.63-3.59 (m, 2H),3.56-3.49 (m, 1H), 3.47-3.39 (m, 3H), 2.62-2.54 (m, 1H), 2.48-2.40 (m,1H), 2.36 (t, J=7.4 Hz, 2H), 2.26 (t, J=7.6 Hz, 2H), 2.19-2.08 (m, 2H),1.65 (quin, J=7.4 Hz, 4H), 1.39-1.24 (m, 25H).

(S)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid

Step 1: Preparation of 18-(tert-butoxy)-18-oxooctadecanoic acid

OCTADECANEDIOIC ACID (7.5 g, 23.85 mmol) was suspended in Toluene (42.6ml) and the mixture was heated to reflux.1,1-di-tert-butoxy-N,N-dimethylmethanamine (15.33 ml, 63.9 mmol) wasadded dropwise over 30 min. The mixture was reflux overnight. Thesolvent was removed in vacuo at 50° C. and the crude material wassuspended in CH₂Cl₂/EtOAc (110 mL. 1:1) and stirred for 15 min. Thesolids were removed by filtration and washed with CH₂Cl₂ (40 mL). Thefiltration was evaporated in vacuo. The crude product was purified byflash chromatography (300 g, SG, 100% CH2Cl2 1000 mL first and then 0 to25% acetone/CH₂Cl₂, 2000 mL) to get 18-(tert-butoxy)-18-oxooctadecanoicacid (3.82 g, 10.31 mmol, 43.2% yield).

Analysis condition D: Retention time=2.85 min; ESI-MS(+) m/z 297.3[M−OC(CH₃)_(3];) ¹H NMR (500 MHz, METHANOL-d₄) δ 2.29 (t, J=7.5 Hz, 2H),2.22 (t, J=7.3 Hz, 2H), 1.67-1.53 (m, 4H), 1.46 (s, 9H), 1.31 (m, 24H).

Step 2: Preparation of (S)-methyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-azidobutanoate

To a mixture of (2S)—N-FMOC-4-AZIDO-BUTANOIC ACID (1.0 g, 2.73 mmol) inMeOH (4.21 ml)/CH₂Cl₂ (12.64 ml) was added (TRIMETHYLSILYL)DIAZOMETHANEin diethyl ether (2.047 ml, 4.09 mmol). The resulting mixture wasstirred at rt for 2 h. The mixture was concentrated to get (S)-methyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-azidobutanoate, whichwas used as is in the next step. ¹H NMR (500 MHz, METHANOL-d₄) δ 7.81(d, J=7.6 Hz, 2H), 7.73-7.63 (m, 2H), 7.47-7.22 (m, 4H), 4.41 (d, J=6.7Hz, 2H), 4.35-4.17 (m, 2H), 3.82-3.70 (m, 3H), 3.49-3.34 (m, 2H),2.21-2.04 (m, 1H), 1.97-1.80 (m, 1H).

Step 3: Preparation of (S)-methyl4-azido-2-((tert-butoxycarbonyl)amino)butanoate

The mixture of (S)-methyl2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-azidobutanoate (1.038 g,2.73 mmol) and diethylamine (4.0 mL, 38.3 mmol) in CH₂Cl₂ (4 mL) wasstirred at rt for 2 h. LCMS showed disappearance of S.M. and productformation along with FMOC related peaks. Concentrated and the resultingproduct was used as is in the next step. ¹H NMR (500 MHz, METHANOL-d4) δ3.85-3.69 (m, 3H), 3.63-3.52 (m, 1H), 3.51-3.40 (m, 2H), 2.05-1.91 (m,1H), 1.88-1.76 (m, 1H).

Step 4: Preparation of (S)-tert-butyl18-((4-azido-1-methoxy-1-oxobutan-2-yl)amino)-18-oxooctadecanoate

The mixture of (S)-methyl 2-amino-4-azidobutanoate (0.432 g, 2.73 mmol),18-(tert-butoxy)-18-oxooctadecanoic acid (1.012 g, 2.73 mmol), DIPEA(1.907 ml, 10.92 mmol) and HBTU (1.035 g, 2.73 mmol) in THF (27.3 ml)was stirred at rt overnight. The resulting crude product was purified byBiotage (silic gel, 300 g, 0 to 10% acetone/CH₂Cl₂) to get(S)-tert-butyl18-((4-azido-1-methoxy-1-oxobutan-2-yl)amino)-18-oxooctadecanoate (1.37g, 2.68 mmol, 98% yield). Analysis condition D: Retention time=2.87 min;ESI-MS(+) m/z 533.3 (M+Na)⁺; ¹H NMR (500 MHz, METHANOL-d₄) δ 4.54 (dd,J=9.2, 5.0 Hz, 1H), 3.78-3.70 (m, 3H), 3.49-3.37 (m, 2H), 2.30-2.19 (m,2H), 2.15-2.04 (m, 1H), 1.97-1.91 (m, 1H), 1.91-1.83 (m, 2H), 1.68-1.53(m, 4H), 1.46 (s, 9H), 1.31 (br. s., 24H).

Step 5: Preparation of(S)-4-azido-2-(18-(tert-butoxy)-18-oxooctadecanamido)butanoic acid

(S)-tert-Butyl18-((4-azido-1-methoxy-1-oxobutan-2-yl)amino)-18-oxooctadecanoate (1.37g, 2.68 mmol), was dissolved in THF (13.41 ml) followed by the additionof lithium hydroxide (0.321 g, 13.41 mmol) and water (13.41 ml). Thereaction was stirred at rt overnight. Concentrated the reaction mixtureto dryness. The resulting(S)-4-azido-2-(18-(tert-butoxy)-18-oxooctadecanamido)butanoic acid wasused as is in the next step. Analysis condition D: Retention time=2.62min; ESI-MS(+) m/z 497.4 (M+H)⁺.

Step 6: Preparation of(S)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid

The mixture of(S)-4-azido-2-(18-(tert-butoxy)-18-oxooctadecanamido)butanoic acid(1.332 g, 2.68 mmol) and TFA (2.89 ml, 37.5 mmol) in DCM (20 ml) wasstirred at rt for 2 h. The resulting crude product was purified byPrep-HPLC (Solvent A=10% MeOH—90% H₂O—0.1% TFA, Solvent B=90% MeOH—10%H2O—0.1% TFA. Column: PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40ml/min, 50-100% B, 10 min and stop at 12 min) to obtain(S)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid (451 mg,1.024 mmol, 38.2% yield). Analysis condition D: Retention time=2.42 min;ESI-MS(+) m/z 441.2 (M+H)⁺; ¹H NMR (500 MHz, METHANOL-d₄) δ 4.51 (dd,4.8 Hz, 1H), 3.51-3.36 (m, 3H), 2.37-2.22 (m, 4H), 2.19-2.08 (m, 1H),1.99-1.88 (m, 1H), 1.68-1.54 (m, 4H), 1.40-1.22 (m, 24H).

(S)-1-azido-39-carboxy-37,41-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,40-diazaoctapentacontan-58-oicAcid

Step 1: Preparation of 18-(tert-butoxy)-18-oxooctadecanoic acid

OCTADECANEDIOIC ACID (7.5 g, 23.85 mmol) was suspended in Toluene (42.6ml) and the mixture was heated to reflux.1,1-di-tert-butoxy-N,N-dimethylmethanamine (15.33 ml, 63.9 mmol) wasadded drop-wise over 30 min. The mixture was reflux overnight. Thesolvent was removed in vacuo at 50° C. and the crude material wassuspended in CH₂Cl₂/EtOAc (110 mL. 1:1) and stirred for 15 min. Thesolids were removed by filtration and washed with CH₂Cl₂ (40 mL). Thefiltration was evaporated in vacuo. The crude product was purified byflash chromatography (SG, 0 to 25% acetone/CH₂Cl₂) to get18-(tert-butoxy)-18-oxooctadecanoic acid (3.95 g, 10.66 mmol, 44.7%yield). Analysis condition D: Retention time=5.04 min; ESI-MS(+) m/z297.3 [M−OC(CH₃)_(3];) ¹H NMR (500 MHz, METHANOL-d₄) δ 2.29 (t, J=7.5Hz, 2H), 2.22 (t, J=7.4 Hz, 2H), 1.67-1.53 (m, 4H), 1.50-1.42 (m, 9H),1.40-1.25 (m, 24H).

Step 2: Preparation of 1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl)octadecanedioate

DCC (5.11 ml, 5.11 mmol) was added to a solution of18-(tert-butoxy)-18-oxooctadecanoic acid (1.72 g, 4.64 mmol) and1-hydroxypyrrolidine-2,5-dione (0.588 g, 5.11 mmol) in DMF (48 mL). Themixture was stirred at rt overnight. The mixture was filtered andconcentrated to get 1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl)octadecanedioate which was used as is in the next step.

Step 3: Preparation of(S)-4-(tert-butoxy)-3-(18-(tert-butoxy)-18-oxooctadecanamido)-4-oxobutanoicacid

Water (6.74 ml) was added to a mixture of(S)-3-amino-4-(tert-butoxy)-4-oxobutanoic acid (1.122 g, 5.93 mmol),1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl) octadecanedioate (2.52 g,5.39 mmol), SODIUM BICARBONATE (0.543 g, 6.47 mmol) in THF (20.21 ml).The resulting clear solution was stirred at rt for 4 h. All THF wasremoved, HCl (7.01 ml, 7.01 mmol) was added and the pH was adjusted to2-3 at 0° C. The resulting suspension was extracted with CH₂Cl₂ (×3),The organic layer was concentrated. The resulting product was used asis. Analysis condition D: Retention time=2.83 min; ESI-MS(+) m/z 542.3(M+H)⁺; ¹H NMR (500 MHz, METHANOL-d₄) δ 4.64 (t, J=6.1 Hz, 1H),2.83-2.67 (m, 2H), 2.29-2.18 (m, 2H), 1.91-1.81 (m, 1H), 1.78-1.67 (m,1H), 1.67-1.53 (m, 4H), 1.53-1.39 (m, 18H), 1.39-1.26 (m, 24H).

Step 4: Preparation of (S)-tert-butyl1-azido-39-(tert-butoxycarbonyl)-37,41-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,40-diazaoctapentacontan-58-oate

To a solution of(S)-4-(tert-butoxy)-3-(18-(tert-butoxy)-18-oxooctadecanamido)-4-oxobutanoicacid (200 mg, 0.369 mmol) in DMF (3692 μl) was added Hunig's Base (193μl, 1.108 mmol) and HATU (281 mg, 0.738 mmol).35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontan-1-amine(211 mg, 0.369 mmol) was then added, and the solution stirred at rt for3 h. The resulting product was used as is. Analysis condition D:Retention time=2.79 min; ESI-MS(+) m/z 1194.7 (M+H)⁺.

Step 5: Preparation of(S)-1-azido-39-carboxy-37,41-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,40-diazaoctapentacontan-58-oicAcid

The mixture of (S)-tert-butyl1-azido-39-(tert-butoxycarbonyl)-37,41-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,40-diazaoctapentacontan-58-oate(404 mg, 0.369 mmol) and TFA (2 mL, 26.0 mmol) in DCM (5 mL) was stirredat rt for 2 h. The resulting crude product was purified by Prep-HPLC(Solvent A=10% MeOH—90% H₂O—0.1% TFA, Solvent B=90% MeOH—10% H₂O—0.1%TFA. Column: PHENOMENEX LUNA 30×150 mm, S10, Flow rate: 40 ml/min,50-100% B, 12 min and stop at 13 min) to obtain(S)-1-azido-39-carboxy-37,41-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,40-diazaoctapentacontan-58-oicacid (128 mg, 0.130 mmol, 35.3% yield) (4 steps yield). Analysiscondition D: Retention time=2.44 min; ESI-MS(+) m/z 982.5 (M+H)⁺.

(S)-1-azido-15-carboxy-13,17-dioxo-3,6,9-trioxa-12,16-diazatetratriacontan-34-oicacid

Step 1: Preparation of 18-(tert-butoxy)-18-oxooctadecanoic acid

OCTADECANEDIOIC ACID (7.5 g, 23.85 mmol) was suspended in toluene (42.6ml) and the mixture was heated to reflux.1,1-di-tert-butoxy-N,N-dimethylmethanamine (15.33 ml, 63.9 mmol) wasadded drop-wise over 30 min. The mixture was reflux overnight. Thesolvent was removed in vacuo at 50° C. and the crude material wassuspended in CH₂Cl₂/EtOAc (110 mL. 1:1) and stirred for 15 min. Thesolids were removed by filtration and washed with CH₂Cl₂ (40 mL). Thefiltration was evaporated in vacuo. The crude product was purified byflash chromatography (SG, 0 to 25% Acetone/CH₂Cl₂) to get18-(tert-butoxy)-18-oxooctadecanoic acid (3.95 g, 10.66 mmol, 44.7%yield). Analysis condition D: Retention time=5.04 min; ESI-MS(+) m/z297.3 [M−OC(CH₃)₃]; ¹H NMR (500 MHz, METHANOL-d₄) δ 2.29 (t, J=7.5 Hz,2H), 2.22 (t, J=7.4 Hz, 2H), 1.67-1.53 (m, 4H), 1.50-1.42 (m, 9H),1.40-1.25 (m, 24H).

Step 2: Preparation of 1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl)octadecanedioate

DCC (5.11 ml, 5.11 mmol) was added to a solution of18-(tert-butoxy)-18-oxooctadecanoic acid (1.72 g, 4.64 mmol) and1-hydroxypyrrolidine-2,5-dione (0.588 g, 5.11 mmol) in DMF (48 mL). Themixture was stirred at rt overnight. The mixture was filtered andconcentrated to get 1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl)octadecanedioate which was used as is in the next step.

Step 3: Preparation of(S)-4-(tert-butoxy)-3-(18-(tert-butoxy)-18-oxooctadecanamido)-4-oxobutanoicacid

Water (6.74 ml) was added to a mixture of(S)-3-amino-4-(tert-butoxy)-4-oxobutanoic acid (1.122 g, 5.93 mmol),1-tert-butyl 18-(2,5-dioxopyrrolidin-1-yl) octadecanedioate (2.52 g,5.39 mmol), SODIUM BICARBONATE (0.543 g, 6.47 mmol) in THF (20.21 ml).The resulting clear solution was stirred at rt for 4 h. All THF wasremoved, HCl (7.01 ml, 7.01 mmol) was added and the pH was adjusted to2-3 at 0° C. The resulting suspension was extracted with CH₂Cl₂ (×3),The organic layer was concentrated. The resulting product was used asis. Analysis condition D: Retention time=2.83 min; ESI-MS(+) m/z 542.3(M+H)⁺; ¹H NMR (500 MHz, METHANOL-d₄) δ 4.64 (t, J=6.1 Hz, 1H),2.83-2.67 (m, 2H), 2.29-2.18 (m, 2H), 1.91-1.81 (m, 1H), 1.78-1.67 (m,1H), 1.67-1.53 (m, 4H), 1.53-1.39 (m, 18H), 1.39-1.26 (m, 24H).

Step 4: Preparation of (S)-tert-butyl1-azido-15-(tert-butoxycarbonyl)-13,17-dioxo-3,6,9-trioxa-12,16-diazatetratriacontane-34-oate

To a solution of(S)-4-(tert-butoxy)-3-(18-(tert-butoxy)-18-oxooctadecanamido)-4-oxobutanoicacid (350 mg, 0.646 mmol) in DMF (6460 μl) was added Hunig's Base (338μl, 1.938 mmol) and HATU (491 mg, 1.292 mmol).2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanamine (141 mg, 0.646 mmol) wasthen added, and the solution stirred at rt for 3 h. The resultingproduct was used as is. Analysis condition D: Retention time=2.90 min;ESI-MS(+) m/z 742.5 (M+H)⁺

Step 5: Preparation of(S)-1-azido-15-carboxy-13,17-dioxo-3,6,9-trioxa-12,16-diazatetratriacontane-34-oicAcid

The mixture of (S)-tert-butyl1-azido-15-(tert-butoxycarbonyl)-13,17-dioxo-3,6,9-trioxa-12,16-diazatetratriacontane-34-oate(479 mg, 0.646 mmol) and TFA (2 mL, 26.0 mmol) in DCM (10 mL) wasstirred at rt for 2 h. The resulting crude product was purified byPrep-HPLC (Solvent A=10% MeOH—90% H₂O—0.1% TFA, Solvent B=90% MeOH—10%H2O—0.1% TFA. Column: PHENOMENEX LUNA 30×150 mm, S10, Flow rate: 40ml/min, 50-100% B, 10 min and stop at 12 min) to obtain(S)-1-azido-15-carboxy-13,17-dioxo-3,6,9-trioxa-12,16-diazatetratriacontane-34-oicacid (119 mg, 0.189 mmol, 29.2% yield) (4 steps yield); Analysiscondition D: Retention time=2.44 min; ESI-MS(+) m/z 630.2 (M+H)⁺.

Preparation of INT-1300A

The following peptide was synthesized on a 0.2 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Ser-Bzt-[N-Me]Nle-[N-Me]Nle-Arg-Cys-Gly-[(S)-propargylglycine].After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: watersxbridge c-18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 40-80% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 37.8 mg, and its estimatedpurity by LCMS analysis was 100%. Analysis condition A: Retentiontime=1.56 min; ESI-MS(+) m/z 986.7 (M+2H), most abundant ion; Analysiscondition B: Retention time=2.86 min; ESI-MS(+) m/z 986.7 (M+2H), mostabundant ion.

Preparation of INT-1300B

The following peptide was synthesized on a 0.2 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Ser-Bzt-[N-Me]Nle-[N-Me]Nle-Arg-Cys-Gly-[(S)-azido-Dab].After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 50-90% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 50.4 mg, and its estimated purity by LCMS analysiswas 100%. Analysis condition A: Retention time=1.61 min; ESI-MS(+) m/z1002.2 (M+2H), most abundant ion; Analysis condition B: Retentiontime=2.82 min; ESI-MS(+) m/z 1002.2 (M+2H), most abundant ion.

Preparation of INT-1300C

The following peptide was synthesized on a 0.1 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Ser-Bzt-[N-Me]Nle-[N-Me]Nle-Arg-Cys-Gly-Gly-[(S)-azido-Dab];After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 45-85% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 9.3 mg, and its estimated purity by LCMS analysis was100%. Analysis condition A: Retention time=1.60 min; ESI-MS(+) m/z1030.7 (M+2H), most abundant ion; Analysis condition B: Retentiontime=2.83 min; ESI-MS(+) m/z 1030.6 (M+2H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 1029.9931 (M+2H) Found: 1029.9898 (M+2H).

Preparation of INT-1300V

The following peptide was synthesized on a 0.1 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure, and italicized residues were coupled with a 30 min singlecoupling.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoic acid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[(S)-propargylglycine];where the (S) propargylglycine was incorporated onto 2-chlorotritylresin. After deprotection and cyclization according to the proceduresabove, the compound was purified as follows: The crude material waspurified via preparative LC/MS with the following conditions: Column:XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95methanol:water with 10-mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10-mM ammonium acetate; Gradient: 45-85% B over 30minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 16.4 mg, and its estimatedpurity by LCMS analysis was 96%. Analysis condition A: Retentiontime=1.49 min; ESI-MS(+) m/z 992.3 (M+2H), most abundant ion. Analysiscondition B: Retention time=3.02 min; ESI-MS(+) m/z 992.3 (M+2H), mostabundant ion; ESI-HRMS(+) m/z: Calculated: 991.9953 (M+2H) Found:991.9926 (M+2H).

Preparation of INT-1300W

The following peptide was synthesized on a 0.2 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure, and italicized residues were coupled with a 30 min singlecoupling.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[(S)-propargylglycine]. Afterdeprotection and cyclization according to the procedures above, thecompound was purified as follows: The crude material was purified viapreparative LC/MS with the following conditions: Column: XBridge C18,19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 50-90% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. Thematerial was further purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 58.8 mg, and itsestimated purity by LCMS analysis was 97%. Analysis condition A:Retention time=1.55 min; ESI-MS(+) m/z 991.9 (M+2H), most abundant ion;Analysis condition B: Retention time=3.11 min; ESI-MS(+) m/z 991.8(M+2H), most abundant ion.

Preparation of INT-1300X

The following peptide was synthesized on a 0.2 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure, and italicized residues were coupled with a 30 min singlecoupling.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Ser-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[(S)-propargylglycine]; Afterdeprotection and cyclization according to the procedures above, thecompound was purified as follows: The crude material was purified viapreparative LC/MS with the following conditions: Column: XBridge C18,19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 39.9 mg, and its estimated purity by LCMSanalysis was 99%. Analysis condition A: Retention time=1.51 min;ESI-MS(+) m/z 985.2 (M+2H), most abundant ion.

Analysis condition B: Retention time=2.62 min; ESI-MS(+) m/z 985.4(M+2H), most abundant ion ESI-HRMS(+) m/z: Calculated: 984.9875 (M+2H)

Found: 984.9877 (M+2H).

Preparation of INT-1300Y

The following peptide was synthesized on a 0.4 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure, and italicized residues were coupled with a 30 min singlecoupling.ClAc-Tyr-[N-Me]Ala-Asp-Pro-His-Lys-Hyp-Trp-Lys-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Glu-Cys-Gly-[(S)-propargylglycine]; where the(S) propargylglycine was incorporated onto 2-chlorotrityl resin. Afterdeprotection and cyclization according to the procedures above, thecompound was purified as follows: The crude material was purified viapreparative LC/MS with the following conditions: Column: XBridge C18,19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 35-75% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. Thematerial was further purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 0-40% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 56.8 mg, and itsestimated purity by LCMS analysis was 100%. Analysis condition A:Retention time=1.06 min; ESI-MS(+) m/z 1047.8 (M+2H), most abundant ion;Analysis condition B: Retention time=2.19 min; ESI-MS(+) m/z 1048.0(M+2H), most abundant ion; ESI-HRMS(+) m/z:

Calculated: 1047.4931 (M+2H); Found: 1047.4899 (M+2H).

Preparation of INT-130AA

The following peptide was synthesized on a 0.8 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure, and italicized residues were coupled with a 30 min singlecoupling.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-Gly-[(S)-propargylglycine]; wherethe (S) propargylglycine was incorporated onto 2-chlorotrityl resin.After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: CSH C18,30×150 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with0.1% trifluoroacetic acid; Gradient: 10-50% B over 30 minutes, then a7-minute hold at 100% B; Flow: 50 mL/min. The sample was divided into 6injections at a concentration of 130 umol per injection. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 102.7 mg, and its estimatedpurity by LCMS analysis was 99%. Analysis condition A: Retentiontime=1.69 min; ESI-MS(+) m/z 1021.1 (M+2H), most abundant ion; Analysiscondition G: Retention time=1.56 min; ESI-MS(+) m/z 1021.3 (M+2H), mostabundant ion; ESI-HRMS(+) m/z: Calculated: 1020.5060 (M+2H) Found:1020.5045 (M+2H).

Preparation of INT-130AB

The following peptide was synthesized on a 0.8 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure, and italicized residues were coupled with a 30 min singlecoupling.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-Gly-Gly-[(S)-propargylglycine];where the (S) propargylglycine was incorporated onto 2-chlorotritylresin. After deprotection and cyclization according to the proceduresabove, the compound was purified as follows: The crude material waspurified via preparative LC/MS with the following conditions: Column:Waters CSH C18, 30×150 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile:water with 0.1% trifluoroacetic acid; Gradient: 5-45% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 132.1 mg, and itsestimated purity by LCMS analysis was 98%. Analysis condition A:Retention time=1.47 min; Analysis condition B: Retention time=1.51 min;ESI-MS(+) m/z 1050.3 (M+2H), most abundant ion; ESI-HRMS(+) m/z:Calculated: 1049.0168 (M+2H)

Found: 1049.0156 (M+2H).

Preparation of INT-130AD

The following peptide was synthesized on a 0.4 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure, and italicized residues were coupled with a 30 min singlecoupling.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Glu-Cys-Gly-[Pra]; where the propargylglycinewas incorporated onto 2-chlorotrityl resin. After deprotection andcyclization according to the procedures above, the compound was purifiedas follows: The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters CSH C18, 30×150 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 0.1%trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1%trifluoroacetic acid; Gradient: 5-45% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 50 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 46.7 mg, and its estimated purity by LCMS analysiswas 97%. Analysis condition A: Retention time=1.31 min; ESI-MS(+) m/z1001.3 (M+2H), most abundant ion; Analysis condition G: Retentiontime=2.19 min; ESI-MS(+) m/z 1000.2 (M+2H); ESI-HRMS(+) m/z: Calculated:999.9746 (M+2H)

Found: 999.9723 (M+2H).

Preparation of INT-130AE

The following peptide was synthesized on a 0.4 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure, and italicized residues were coupled with a 30 min singlecoupling.ClAc-Tyr-[N-Me]Ala-Asp-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Glu-Cys-Gly-[Pra]; where the propargylglycinewas incorporated onto 2-chlorotrityl resin. After deprotection andcyclization according to the procedures above, the compound was purifiedas follows: The crude material was purified via preparative LC/MS withthe following conditions: Column: Waters CSH C18, 30×150 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 0.1%trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1%trifluoroacetic acid; Gradient: 5-45% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 50 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 51.0 mg, and its estimated purity by LCMS analysiswas 96%. Analysis condition A: Retention time=1.23 min; ESI-MS(+) m/z1001.2 (M+2H), most abundant ion. Analysis condition G: Retentiontime=1.27 min; ESI-MS(+) m/z 1000.9 (M+2H); ESI-HRMS(+) m/z: Calculated:1000.4666 (M+2H)

Found: 1000.4646 (M+2H).

Preparation of INT-130AF

The following peptide was synthesized on a 0.8 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure, and italicized residues were coupled with a 30 min singlecoupling.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-[Pra]; where the propargylglycine wasincorporated onto 2-chlorotrityl resin. After deprotection andcyclization according to the procedures above, the compound was purifiedas follows: The crude material was purified via preparative HPLC withthe following conditions: Column: Phenomenex Gemini NX-C18, 50×250 mm,5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.05%trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.05%trifluoroacetic acid; Gradient: 10-50% B over 30 minutes, then a5-minute hold at 100% B; Flow: 125 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 152.1 mg, and its estimated purity by LCMSanalysis was 100%. Analysis condition A: Retention time=1.56 min;ESI-MS(+) m/z 964.1 (M+2H); Analysis condition G: Retention time=1.39min; ESI-MS(+) m/z 965.2 (M+2H), most abundant ion; ESI-HRMS(+) m/z:Calculated: 963.4846 (M+2H)

Found: 963.4825 (M+2H).

Preparation of INT-130AG

The following peptide was synthesized on a 0.8 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure, and italicized residues were coupled with a 30 min singlecoupling.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Glu-Cys-[Pra]; where the propargylglycine wasincorporated onto 2-chlorotrityl resin. After deprotection andcyclization according to the procedures above, the compound was purifiedas follows: The crude material was purified via preparative LC/MS withthe following conditions: Column: Phenomenex Gemini NX-C18, 50×250 mm,5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.05%trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.05%trifluoroacetic acid; Gradient: 5-45% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 125 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 154.1 mg, and its estimated purity by LCMS analysiswas 100%. Analysis condition A: Retention time=1.28 min Analysiscondition G: Retention time=1.25 min; ESI-HRMS(+) m/z:

Calculated: 971.4638 (M+2H) Found: 971.4620 (M+2H).

Preparation of INT-130AH

The following peptide was synthesized on a 0.8 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure, and italicized residues were coupled with a 30 min singlecoupling.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Asp-Cys-[Pra]; where the propargylglycine wasincorporated onto 2-chlorotrityl resin. After deprotection andcyclization according to the procedures above, the compound was purifiedas follows: The crude material was purified via preparative HPLC withthe following conditions: Column: Phenomenex Gemini NX-C18, 50×250 mm,5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.05trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.05%trifluoroacetic acid; Gradient: 5-45% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 125 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 147.7 mg, and its estimated purity by LCMS analysiswas 100%. Analysis condition A: Retention time=1.29 min; ESI-MS(+) m/z965.3 (M+2H); Analysis condition G: Retention time=1.24 min; ESI-MS(+)m/z 964.9 (M+2H); ESI-HRMS(+) m/z: Calculated: 964.4560 (M+2H) Found:964.4535 (M+2H).

Preparation of INT-130AI

The following peptide was synthesized on a 0.1 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure, whereas the italicized coupling was a single, 30 mincoupling.ClAc-Tyr-hPro-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-mNle-mNle-Leu-Cys-Gly-[(S)-propargylglycine]. After deprotectionand cyclization according to the procedures above, the compound waspurified as follows: The crude material was purified via preparativeLC/MS with the following conditions: Column: Phenomenex Gemini NX-C18,50×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with0.1% trifluoroacetic acid; Gradient: 10-50% B over 30 minutes, then a5-minute hold at 100% B; Flow: 100 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 158.7 mg, and its estimated purity by LCMSanalysis was 98%. Analysis condition A: Retention time=1.556 min;ESI-MS(+) m/z 1005.1 (M+2H), most abundant ion; Analysis condition G:Retention time=1.382 min; ESI-MS(+) m/z 1005.3 (M+2H), most abundantion; ESI-HRMS(+) m/z: Calculated: 1005.0031 (M+2H) Found: 1005.0015(M+2H).

Preparation of INT-130AJ

The following peptide was synthesized on a 0.1 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-hPro-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-mNle-mNle-Glu-Cys-Gly-[(S)-propargylglycine]. After deprotectionand cyclization according to the procedures above, the compound waspurified as follows: The crude material was purified via preparativeLC/MS with the following conditions: Column: Phenomenex Gemini NX-C18,50×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with0.1% trifluoroacetic acid; Gradient: 5-45% B over 30 minutes, then a5-minute hold at 100% B; Flow: 100 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 107.7 mg, and its estimated purity by LCMSanalysis was 100%. Analysis condition A: Retention time=1.281 min;ESI-MS(+) m/z 1013.1 (M+2H), most abundant ion; Analysis condition G:Retention time=1.282 min; ESI-MS(+) m/z 1013.2 (M+2H), most abundantion; ESI-HRMS(+) m/z: Calculated: 1012.9824 (M+2H) Found: 1012.9797(M+2H).

Preparation of INT-130AK

The following peptide was synthesized on a 0.1 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-hPro-Asn-Pro-DapMe-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-mNle-mNle-Leu-Cys-Gly-[(S)-propargylglycine]. After deprotectionand cyclization according to the procedures above, the compound waspurified as follows: The crude material was purified via preparativePrep HPLC with the following conditions: Column: Phenomenex GeminiNX-C18, 50×250 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile:water with 0.1% trifluoroacetic acid; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 125 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 241.9 mg, and itsestimated purity by LCMS analysis was 98%. Analysis condition A:Retention time=1.555 min; ESI-MS(+) m/z 1012.1 (M+2H), most abundantion; Analysis condition G: Retention time=1.396 min; ESI-MS(+) m/z1012.2 (M+2H), most abundant ion; ESI-HRMS(+) m/z: Calculated: 1012.0109(M+2H) Found: 1012.0089 (M+2H).

Preparation of INT-130AL

The following peptide was synthesized on a 0.1 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-hPro-Asn-Pro-DapMe-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-mNle-mNle-Glu-Cys-Gly-[(S)-propargylglycine]. After deprotectionand cyclization according to the procedures above, the compound waspurified as follows: The crude material was purified via preparativeLC/MS with the following conditions: Column: Phenomenex Gemini NX-C18,50×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with0.1% trifluoroacetic acid; Gradient: 5-45% B over 30 minutes, then a7-minute hold at 100% B; Flow: 100 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 218.0 mg, and its estimated purity by LCMSanalysis was 100%. Analysis condition A: Retention time=1.316 min;ESI-MS(+) m/z 1020.2 (M+2H), most abundant ion; Analysis condition G:Retention time=1.294 min; ESI-MS(+) m/z 1020.2 (M+2H), most abundantion; ESI-HRMS(+) m/z: Calculated: 1019.9902 (M+2H) Found: 1019.9881(M+2H).

Preparation of Example 13051

Intermediate 1300B (10 mg, 4.99 μmol) andN-(prop-2-yn-1-yl)tetradecanamide (3.98 mg, 0.015 mmol) were reacted asin the general triazole formation procedure to afford crude product. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×mm, 5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 35-85% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The material was further purified viapreparative LC/MS with the following conditions: Column: XBridge C18,19×mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with 10-mMammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 60-100% B over 20 minutes, then a 10-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 2.5 mg, and its estimated purity by LCMS analysis was100%. Analysis condition A: Retention time=2.32 min; ESI-MS(+) m/z1134.9 (M+2H); Analysis condition B: Retention time=3.35 min; ESI-MS(+)m/z 1135.1 (M+2H), most abundant ion; ESI-HRMS(+) m/z: Calculated:1134.1026 (M+2H)

Found: 1134.1028 (M+2H).

Preparation of Example 13052

Intermediate 1300B (8.5 mg, 4.25 μmol) and2-(2-(2-methoxyethoxy)ethoxy)-N-(prop-2-yn-1-yl)acetamide (2.74 mg,0.013 mmol) were reacted as in the general triazole formation procedureto afford crude product. The crude material was purified via preparativeLC/MS with the following conditions: Column: XBridge C18, 19×200 mm,5-μm particles; Mobile Phase A: 5:95 methanol:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammoniumacetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at100% B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 6.3 mg, and its estimated purity by LCMS analysis was 100%. Analysiscondition A: Retention time=1.56 min; ESI-MS(+) m/z 1109.9 (M+2H);Analysis condition B: Retention time=2.81 min; ESI-MS(+) m/z 1109.8(M+2H), most abundant ion.

Preparation of Example 13121

Intermediate 130AB (26.1 mg, 12 μmol) and(S)-4-azido-2-palmitamidobutanoic acid (13.7 mg, 0.031 mmol) werereacted as in the general triazole formation procedure to afford crudeproduct. The reaction solution was diluted slightly in MeOH and purifiedby prep HPLC (2 injections): 30×100 mm HPLC Phenomenex Luna 5 μm 10 to100% A:B over 15 min, 3 min at 100% B (A is 90:10 water:CH₃CN w/0.1%TFA; B is 10:90 water:CH₃CN w/0.1% TFA)). The yield of the product was11.7 mg, and its estimated purity by LCMS analysis was 99%. Analysiscondition H: Retention time=10.92 min

Analysis condition I: Retention time=9.94 min; ESI-HRMS(+) m/z:

Calculated: 1269.1667 (M+2H) Found: 1269.1648 (M+2H).

Preparation of Example 13122

Intermediate 130AA (25.5 mg, 13 μmol) and(S)-4-azido-2-palmitamidobutanoic acid (13.8 mg, 0.031 mmol) werereacted as in the general triazole formation procedure to afford crudeproduct. The reaction solution was diluted slightly in MeOH and purifiedby prep HPLC (2 injections): 30×100 mm HPLC Phenomenex Luna 5 μm 10 to100% A:B over 15 min, 3 min at 100% B (A is 90:10 water:CH₃CN w/0.1%TFA; B is 10:90 water:CH₃CN w/0.1% TFA)). The yield of the product was8.1 mg, and its estimated purity by LCMS analysis was 94%. Analysiscondition I: Retention time=10.06 min

Analysis condition J: Retention time=8.63 min; ESI-HRMS(+) m/z:

Calculated: 1240.6560 (M+2H) Found: 1240.6546 (M+2H).

Preparation of Example 13123

Intermediate 130AA (41.3 mg, 20 μmol) and(S)-1-azido-28-carboxy-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oicacid (24.9 mg, 0.030 mmol) were reacted as in the general triazoleformation procedure to afford crude product. The reaction solution wasdiluted slightly in MeOH and purified by prep HPLC (2 injections):30×100 mm HPLC Phenomenex Luna 5 μm 10 to 100% A:B over 15 min, 3 min at100% B (A is 90:10 water:CH₃CN w/0.1% TFA; B is 10:90 water:CH₃CN w/0.1%TFA)). The yield of the product was 26.2 mg, and its estimated purity byLCMS analysis was 94%. Analysis condition I: Retention time=9.54 min

Analysis condition J: Retention time=8.13 min; ESI-HRMS(+) m/z:

Calculated: 1430.2663 (M+2H) Found: 1430.2656 (M+2H).

Preparation of Example 13124

Intermediate 130AB (40.7 mg, 19 μmol) and(S)-1-azido-28-carboxy-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oicacid (23.9 mg, 0.029 mmol) were reacted as in the general triazoleformation procedure to afford crude product. The reaction solution wasdiluted slightly in MeOH and purified by prep HPLC (2 injections):30×100 mm HPLC Phenomenex Luna 5 μm 10 to 100% A:B over 15 min, 3 min at100% B (A is 90:10 water:CH₃CN w/0.1% TFA; B is 10:90 water:CH₃CN w/0.1%TFA)). The yield of the product was 24.3 mg, and its estimated purity byLCMS analysis was 95%. Analysis condition I: Retention time=9.49 min;Analysis condition J: Retention time=8.08 min; ESI-HRMS(+) m/z:

Calculated: 1458.7770 (M+2H) Found: 1458.7743 (M+2H).

Preparation of Example 13125

Intermediate 130AD (22.1 mg, 11 μmol) and(S)-4-azido-2-palmitamidobutanoic acid (6.1 mg, 0.014 mmol) were reactedas in the general triazole formation procedure to afford crude product.The reaction solution was diluted slightly in MeOH and purified by prepHPLC (2 injections): 30×100 mm HPLC Phenomenex Luna 5 μm 10 to 100% A:Bover 15 min, 3 min at 100% B (A is 90:10 water:CH₃CN w/0.1% TFA; B is10:90 water:CH₃CN w/0.1% TFA)). The yield of the product was 9.5 mg, andits estimated purity by LCMS analysis was 90%. Analysis condition I:Retention time=9.24 min; Analysis condition J: Retention time=8.00 min;ESI-HRMS(+) m/z: Calculated: 1220.1245 (M+2H)

Found: 1220.1208 (M+2H).

Preparation of Example 13126

Intermediate 130AE (24.5 mg, 12 μmol) and(S)-4-azido-2-palmitamidobutanoic acid (6.8 mg, 0.015 mmol) were reactedas in the general triazole formation procedure to afford crude product.The reaction solution was diluted slightly in MeOH and purified by prepHPLC (2 injections): 30×100 mm HPLC Phenomenex Luna 5 μm 10 to 100% A:Bover 15 min, 3 min at 100% B (A is 90:10 water:CH₃CN w/0.1% TFA; B is10:90 water:CH₃CN w/0.1% TFA)). The yield of the product was 10.3 mg,and its estimated purity by LCMS analysis was 95%. Analysis condition I:Retention time=9.36 min

Analysis condition J: Retention time=8.15 min; ESI-HRMS(+) m/z:

Calculated: 1220.6165 (M+2H) Found: 1220.6133 (M+2H).

Preparation of Example 13129

Intermediate 130AF (35 mg, 18.0 μmol) and(S)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid (10.01mg, 0.023 mmol), were reacted as in the general triazole formationprocedure above to afford crude product. The crude product was purifiedby Prep-HPLC (Column: Phenomenex Luna C18 30×100 mm Sum, Solvent A=90:10H₂O:ACN 0.1% TFA, Solvent B=10:90 H₂O:CAN 0.1% TFA. Flow rate: 40ml/min, 10-100% B. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 11.7 mg, and its estimated purity by LCMS analysis was 96%. Analysiscondition C: Retention time=1.162 min; ESI-MS(+) m/z 1185.50 (M+2H),most abundant ion.

Preparation of Example 13130

Intermediate 130AG (40.65 mg, 21.0 μmol) and(S)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid (11.53mg, 0.026 mmol), were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 40-80% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 4.9 mg, and its estimated purity by LCMS analysis was93%. Analysis condition A: Retention time=1.541 min; ESI-MS(+) m/z1191.5 (M+2H), most abundant ion; Analysis condition G: Retentiontime=1.682 min; ESI-MS(+) m/z 1192.2 (M+2H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 1191.6138 (M+2H)

Found: 1191.6106 (M+2H).

Preparation of Example 13131

Intermediate 130AH (40.37 mg, 21.0 μmol) and(S)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid (11.53mg, 0.026 mmol), were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 40-80% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 2.1 mg, and its estimated purity by LCMS analysis was95%. Analysis condition A: Retention time=1.815 min; Analysis conditionK: Retention time=1.684 min; ESI-MS(+) m/z 1185.2 (M+2H), most abundantion; ESI-HRMS(+) m/z:

Calculated: 1184.6059 (M+2H) Found: 1184.6031 (M+2H).

Preparation of Example 13132

Intermediate 130AI (46.4 mg, 23.0 μmol) and(S)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid (11.19mg, 0.025 mmol), were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: Waters CSHC18, 19×mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with0.1% trifluoroacetic acid; Gradient: 30-70% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 7.2 mg, and its estimated purity by LCMSanalysis was 100%. Analysis condition G: Retention time=1.888 min;ESI-MS(+) m/z 817.9 (M+3H), most abundant ion; ESI-HRMS(+) m/z:Calculated: 1225.1531 (M+2H)

Found: 1225.1521 (M+2H).

Preparation of Example 13133

Intermediate 130AK (56.5 mg, 28.0 μmol) and(S)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid (13.53mg, 0.031 mmol), were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: Waters CSHC18, 19×mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with0.1% trifluoroacetic acid; Gradient: 30-70% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 9.8 mg, and its estimated purity by LCMSanalysis was 99%. Analysis condition A: Retention time=1.788 min;ESI-MS(+) m/z 1232.2 (M+2H), most abundant ion; Analysis condition G:Retention time=1.843 min; ESI-MS(+) m/z 1233.1 (M+2H), most abundantion; ESI-HRMS(+) m/z: Calculated: 1232.1609 (M+2H) Found: 1232.1579(M+2H).

Cyclization Method B:

All manipulations were performed manually unless noted. The procedure of“Cyclization Method B” describes an experiment performed on a 0.100 mmolscale, where the scale is determined by the amount resin used togenerate the peptide. This scale is not based on a direct determinationof the quantity of peptide used in the procedure. The procedure can bescaled beyond 0.100 mmol scale by adjusting the described volumes by themultiple of the scale. The crude peptide solids were dissolved inMethanol (˜24 mL) containing ˜3 drops of N,N-Diisopropylethylamine. Thebasic (pH>9) solution was then allowed to stand for 18-24 h. To theresulting solution, 1 mL DMSO was added, and the methanol portionevaporated under reduced pressure to afford a concentrated DMSO solutionof the crude, cyclized product. This solution was subjected toreverse-phase HPLC purification to afford the desired cyclic peptide.

Analysis Condition 13A:

Column: X-Select CSH C18, 3.0×150 mm, 3.5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 0.1% TFA; Mobile Phase B: 95:5acetonitrile:water with 0.1% TFA; Gradient: 10-100% B over 15 minutes;Flow: 0.5 mL/min; Detection: UV at 220 nm.

Analysis Condition 13B:

Column: Zorbax Bonus-RP C18, 3.0×150 mm, 3.5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 0.1% TFA; Mobile Phase B: 95:5acetonitrile:water with 0.1% TFA; Gradient: 10-100% B over 15 minutes;Flow: 0.5 mL/min; Detection: UV at 220 nm.

Preparation of 1-tert-butyl 18-(perfluorophenyl) octadecanedioate

To a solution of 18-(tert-butoxy)-18-oxooctadecanoic acid (5.00 g, 13.49mmol) in DMF (54.0 ml) was added pyridine (3.82 ml, 47.2 mmol), followedby pentafluorophenyl trifluoroacetate (5.81 ml, 33.7 mmol). A gelformed, and an additional stir bar was added to the reaction mixture.The mixture was stirred vigorously overnight. The reaction mixture wasfiltered (Buchner funnel/paper) to afford a white solid, which washedwith a small amount of DMF. A nitrogen-rich atmosphere was suckedthrough the filter cake for a few hours to provide 1-tert-butyl18-(perfluorophenyl) octadecanedioate (6.24 g, 11.63 mmol, 86% yield).

Preparation of (S)-1-tert-butyl 5-(perfluorophenyl)2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanedioate

To a solution of(S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (350 mg, 0.630 mmol) in DMF (2519 μl) was added pyridine (178 μl,2.204 mmol), followed by pentafluorophenyl trifluoroacetate (271 μl,1.574 mmol). The vial containing the resulting solution was flushed withnitrogen, and capped overnight. The observed product was observed byLC/MS. The mixture was diluted with water and citric acid, and extracted3 times with EtOAc. The combined organic extracts were dried over MgSO₄,filtered, and concentrated in vacuo. The residue was used as-is forfurther chemistry.

Preparation of(S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanoicacid

To a solution of 1-tert-butyl 18-(perfluorophenyl) octadecanedioate(1.666 g, 3.10 mmol) in DMF (21.71 ml) was added(S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-aminopentanoic acid(1.0 g, 2.82 mmol) and Hunig's Base (1.478 ml, 8.47 mmol). The mixturewas stirred at rt. LC/MS showed the formation of the desired product.The mixture was diluted with an aqueous citric acid solution, and wasextracted 3 times into CH₂Cl₂. The combined organic extracts were driedover MgSO₄, filtered, and concentrated in vacuo.

The bulk material was purified by PREP HPLC: (50×250 mm HPLC Sunfire C1810 μm 0 to 100% A:B over 40 min, 10 min at 100% B (A is 90:10:0.1water:MeOH:TFA; B is 90:10:0.1 MeOH:water:TFA)). Subsequent runs werepurified at 100% B (isocratic) for 15 min. Fractions were pooled,adjusted to neutral pH with Hunig's base, and concentrated in therotovap. The residual aqueous layer was extracted 3 times with EtOAc,filtered, and concentrated in vacuo to afford(S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanoicacid (1.33 g, 1.881 mmol, 66.7% yield).

Preparation of(S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)butanoicacid

To a suspension of 1-tert-butyl 18-(perfluorophenyl) octadecanedioate(1.155 g, 2.152 mmol) in DMF (1.66E+04 μl) was added(S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-aminobutanoic acid(827 mg, 2.430 mmol) and Hunig's Base (1128 μl, 6.46 mmol). The mixturewas stirred vigorously over the weekend. The LC/MS showed the desiredproduct. The mixture was diluted with aqueous citric acid, and extracted3 times into CH₂Cl₂. The combined organic extracts were dried overMgSO₄, filtered, and concentrated in vacuo. The material was taken up ina cloudy solution in MeOH. Filtration through a 0.45 um frit was slow(several filters were used) to give a clear solution. Purified by PREPHPLC: (50×250 mm HPLC Sunfire C18 10 μm 100% B over 14 min, (A is90:10:0.1 water:MeOH:TFA; B is 90:10:0.1 MeOH:water:TFA)). Fractionswere pooled, adjusted to neutral with Hunig's base, and concentrated inthe rotovap. Isolated(S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)butanoicacid (778.42 mg, 1.123 mmol, 52.2% yield).

Preparation of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanamido)butanoicacid

To a solution of crude(S)-1-tert-butyl 5-(perfluorophenyl)2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanedioate (455 mg, 0.63mmol) in DMF (6300 μl) was added Hunig's Base (440 μl, 2.52 mmol), then(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-aminobutanoic acid(214 mg, 0.630 mmol). The mixture was stirred at rt overnight. Themixture was diluted with citric acid and extracted 3 times into EtOAc.The combined organic extracts were washed with brine, dried over MgSO₄,filtered, and concentrated in vacuo. The residue was purified by PREPHPLC: (50×250 mm HPLC Sunfire C18 10 μm 10 to 100% A:B over 30 min, 5min at 100% B (A is 90:10:0.1 water:MeOH:TFA; B is 90:10:0.1MeOH:water:TFA)). The material eluted during the 100% B isocraticsection, so the gradient was not necessary. Fractions were neutralizedwith Hunig's base and concentrated on the speedvac. The residue wastaken up in EtOAc and washed twice with water and once with brine. Theorganic layer was dried over MgSO4, filtered, and concentrated in vacuoto afford(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanamido)butanoicacid (288 mg, 0.328 mmol, 52.1% yield).

Preparation of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanamido)propanoicacid

To a solution of crude(S)-1-tert-butyl 5-(perfluorophenyl)2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanedioate (1163 mg, 1.611mmol) in DMF (1.61E+04 μl) was added Hunig's Base (1125 μl, 6.44 mmol),then (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-aminopropanoicacid (578 mg, 1.772 mmol). The mixture was left to stir at rt overnight.LC/MS showed the formation of product. The mixture was diluted withwater. Citric acid was added, resulting in a white precipitate. Themixture was extracted with EtOAc, and after vigorous shaking the soliddissolved. An emulsion formed, and DCM and salt water were added to aidin the layer separation. The combined layers were passed through a padof celite to aid in emulsion separation, leaving a gooey residue on thecelite. The layers were separated, and the aqueous phase washed withDCM. The combined organic extracts were washed with brine, dried overMgSO4, filtered, and concentrated in vacuo. The residue was purified byPREP HPLC: (50×250 mm HPLC Sunfire C18 10 μm 100% B for 14 min (A is90:10:0.1 water:MeOH:TFA; B is 90:10:0.1 MeOH:water:TFA)). Fractionswere neutralized with Hunig's base and concentrated on the rotovap toafford(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanamido)propanoicacid (940 mg, 1.088 mmol, 67.5% yield).

Preparation of modified chlorotrityl resin 13A

2-Chlorotrityl resin (713 mg, 1.141 mmol) was swelled with CH₂Cl₂ (7mL). A solution of(S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanoicacid (252 mg, 0.356 mmol), Hunig's Base (0.405 mL, 2.317 mmol), and1-chloro-4-methylbenzene (0.021 mL, 0.178 mmol) in CH₂Cl₂ (3.5 mL) wasprepared, analyzed by LC/MS, and added to the swelled resin. The mixturewas shaken at rt, monitoring for disappearance of SM. After ˜45 min, thematerial was almost completely attached to the resin. 13 mL of 9:1MeOH/Hunig's base was added, and the mixture filtered within 1 minute.The resin was rinsed 3 times with DCM (stirring for ˜20 s in betweenwashes). The resin was then shaken with ˜20 mL DMF for 5 min, thenfiltered. This was repeated 2 more times with DMF, then 3 times withDCM. The resin was dried on the fritted funnel with N2 being passedthrough the resin.

Total weight of resin is 0.76 g. 60.06 mg of resin was taken and wasshaken for 1 min with 1 mL of 20% hexafluoroisopropanol in DCM. Filteredand rinsed with more cleavage solution, then DCM. Concentration of thecombined filtrates yielded 11.94 mg (0.01689 mmol) cleaved material.Thus the measured loading was 0.281 meq/g, and the incorporated yieldwas 60%.

Preparation of Modified Chlorotrityl Resin 13B

2-Chlorotrityl resin (1622 mg, 2.60 mmol) was swelled with CH₂Cl₂(1.59E+04 μl). A solution of(S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)butanoicacid (561.95 mg, 0.811 mmol), Hunig's Base (921 μl, 5.27 mmol), and1-chloro-4-methylbenzene (48.0 μl, 0.405 mmol) in CH₂Cl₂ (7951 μl) wasprepared, analyzed by LC/MS, and added to the swelled resin. The mixturewas shaken at rt, monitoring for disappearance of SM. After ˜45 min, thematerial was almost completely attached to the resin. 30 mL of 9:1MeOH/Hunig's base was added, and the mixture filtered within 1 minute.The resin was rinsed 3 times with DCM (stirring for ˜20 s in betweenwashes). The resin was then shaken with ˜20 mL DMF for 5 min, thenfiltered. This was repeated 2 more times with DMF, then 3 times withDCM. The resin was dried on the fritted funnel with N₂ being passedthrough the resin. The total weight of resin was 1.93 g. 76.45 mg ofresin and was shaken for 1 min with 1 mL of 20% hexafluoroisopropanol inDCM. Filtered and rinsed with more cleavage solution, then DCM.Concentration of the combined filtrates yielded 20.67 mg (0.02983 mmol)cleaved material. Thus, the measured loading was 0.39 meq/g, and theincorporated yield is 93%.

Preparation of Modified Chlorotrityl Resin 13C

2-Chlorotrityl resin (656 mg, 1.049 mmol) was swelled with CH₂Cl₂ (6431μl). After 10 min, 186 uL of Hunig's base was added, and the resultingwhite smoke was flushed out of the flask with nitrogen. A solution of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanamido)butanoicacid (288 mg, 0.328 mmol), Hunig's Base (372 μl, 2.132 mmol), and1-chloro-4-methylbenzene (19.42 μl, 0.164 mmol) in CH₂Cl₂ (3215 μl) wasprepared, analyzed by LC/MS, and added to the swelled resin. NOTE: a 0.5mL portion of DMF was added to aid in the dissolution of the startingmaterial. The mixture was shaken at rt, monitoring for disappearance ofSM. The reaction appeared to stall, so another 186 uL of Hunig's basewas added. After an additional 30 min no meaningful progress was seen.131 mg of chlorotrityl resin was added, and again the mixture was shakenat rt overnight. LC/MS still showed SM, but the reaction was terminatedanyway. 30 mL of 9:1 MeOH/Hunig's base was added, and the mixturefiltered within a minute. The resin was rinsed 3 times with DCM(stirring for ˜20 s in between washes). The resin was then shaken with˜20 mL DMF for 5 min, then filtered. This was repeated 2 more times withDMF, then 3 times with DCM. The resin was dried on the fritted funnelwith N₂ being passed through the resin.

The total weight of resin was 954 mg. 49.96 mg of resin was shaken for 1min with 2 mL of 20% hexafluoroisopropanol in DCM. Filtered and rinsedwith more cleavage solution, then DCM. Concentration of the combinedfiltrates yielded 10.16 mg (0.01157 mmol) cleaved material. Thus, themeasured loading was 0.23 meq/g, and the incorporated yield was 67%.

Preparation of modified chlorotrityl resin 13D

2-Chlorotrityl resin (2176 mg, 3.48 mmol) was swelled with CH₂Cl₂(2.13E+04 μl). After 10 min, 186 uL of Hunig's base was added, and theresulting white smoke was flushed out of the flask with nitrogen. Asolution of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((S)-5-(tert-butoxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanamido)propanoicacid (940 mg, 1.088 mmol), Hunig's Base (1235 μl, 7.07 mmol), and1-chloro-4-methylbenzene (64.4 μl, 0.544 mmol) in CH₂Cl₂ (1.07E+04 μl)was prepared, analyzed by LC/MS, and added to the swelled resin. Themixture was shaken at rt, monitoring for disappearance of SM. Thereaction was ˜97% complete at ˜1 h. 100 mL of 9:1 MeOH/Hunig's base wasadded, and the mixture filtered within 1 minute. The resin was rinsed 3times with DCM (stirring for ˜20 s in between washes). The resin wasthen shaken with ˜60 mL DMF for 5 min, then filtered. This was repeated2 more times with DMF, then 3 times with DCM. The resin was dried on thefitted funnel with N₂ being passed through the resin. The total weightof resin was 2.57 g. 85.11 mg of resin was shaken for 1 min with 2 mL of20% hexafluoroisopropanol in DCM. Filtered and rinsed with more cleavagesolution, then DCM. Concentration of the combined filtrates yielded17.51 mg (0.02026 mmol) cleaved material. Thus, the measured loading was0.238 meq/g, and the incorporated yield was 56%.

Preparation of Example 13141

Example 13141 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-Asp(OtBu)-[Modified resin 13A].After deprotection following Global Deprotection procedure B andcyclization according to Cyclization method B, the compound was purifiedas follows: The residue was split and multiple injections were made to a19×250 XSelect CSH Prep C18 5 um OBD column, 20 mL/min, 20 to 65% B over25 min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1%TFA; B is 90% ACN/10% water/0.1% TFA). Conc ammonia was used to adjustthe fractions to pH 7, and the fractions concentrated on the speedvac.Analytical data supported the submission of(6S,13S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-31-((H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,8,15-tetraoxo-2,5,9,14-tetraazahentriacontane-6,13,31-tricarboxylicacid (6.01 mg, 2.242 μmol, 2.242% yield). Analysis condition 13A: rt9.96 min. Analysis condition 13B: rt 8.61 min.; ESI-HRMS(+) m/z:Calculated: 1220.1553 (M+2H) Found: 1220.1573 (M+2H).

Preparation of Example 13142

Example 13142 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-Glu(OtBu)-[Modified resin 13A].After deprotection following Global Deprotection procedure B andcyclization according to Cyclization method B, the compound was purifiedas follows: The residue was split and multiple injections were made to a19×250 XSelect CSH Prep C18 5 um OBD column, 20 mL/min, 20 to 65% B over25 min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1%TFA; B is 90% ACN/10% water/0.1% TFA). Conc. ammonia was used to adjustthe fractions to pH 7, and the fractions concentrated on the speedvac.The residual solid contained a large amount of ammoniumtrifluoroacetate, which was removed by multiple lyophilization cycles toafford(6S,14S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-31-((1H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-25-((1-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,9,16-tetraoxo-2,5,10,15-tetraazadotriacontane-6,14,32-tricarboxylicacid, 2 TFA (3.66 mg, 1.296 μmol, 1.296% yield). Analysis condition 13A:rt 10.00 min. Analysis condition 13B: rt 8.64 min.; ESI-HRMS(+) m/z:Calculated: 1227.1631 (M+2H) Found: 1227.1669 (M+2H).

Preparation of Example 13143

Example 13143 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-Glu(OtBu)-[Modified resin 13B].After deprotection following Global Deprotection procedure B andcyclization according to Cyclization method B, the compound was purifiedas follows: The residue was split and multiple injections were made to a19×250 XSelect CSH Prep C18 5 um OBD column, 20 mL/min, 20 to 65% B over25 min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1%TFA; B is 90% ACN/10% water/0.1% TFA). ACN was removed from thefractions on the rotovap, and the aqueous portion was lyophilized.Analytical data supported the submission of(6S,13S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-31-((1H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-25-((1-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,9,15-tetraoxo-2,5,10,14-tetraazahentriacontane-6,13,31-tricarboxylicacid (5.93 mg, 2.406 μmol, 2.406% yield). Analysis condition 13A: rt9.97 min. Analysis condition 13B: rt 8.61 min.; ESI-HRMS(+) m/z:Calculated: 1220.1553 (M+2H)

Found: 1220.1601 (M+2H).

Preparation of Example 13144

Example 13144 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-Asp(OtBu)-[Modified resin 13B].After deprotection following Global Deprotection procedure B andcyclization according to Cyclization method B, the compound was purifiedas follows: The residue was split and multiple injections were made to a30×150 XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over25 min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1%TFA; B is 90% ACN/10% water/0.1% TFA). ACN was removed from thefractions on the rotovap, and the aqueous portion was lyophilized.Analytical data supported the submission of(6S,12S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-3141H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,8,14-tetraoxo-2,5,9,13-tetraazatriacontane-6,12,30-tricarboxylicacid, 2 TFA (9.57 mg, 3.43 μmol, 3.43% yield). Analysis condition 13A:rt 9.98 min. Analysis condition 13B: rt 8.66 min.; ESI-HRMS(+) m/z:Calculated: 1213.1474 (M+2H) Found: 1213.1513 (M+2H).

Preparation of Example 13145

Example 13145 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-[p-Fluorophenylalanine]-Pip-Asp-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-Asp(OtBu)-[Modified resin 13A].After deprotection following Global Deprotection procedure B andcyclization according to Cyclization method B, the compound was purifiedas follows: The residue was split and multiple injections were made to a30×150 XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over25 min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1%TFA; B is 90% ACN/10% water/0.1% TFA). ACN was removed from thefractions on the rotovap, and the aqueous portion was lyophilized.Analytical data supported the submission of(6S,13S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-3141H-indol-3-yl)methyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-50-(carboxymethyl)-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-7-(4-fluorobenzyl)-35-hydroxy-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,8,15-tetraoxo-2,5,9,14-tetraazahentriacontane-6,13,31-tricarboxylicacid, 2 TFA (10.57 mg, 3.88 μmol). Analysis condition 13A: rt 11.43 min.Analysis condition 13B: rt 9.57 min.; ESI-HRMS(+) m/z: Calculated:814.7658 (M+3H)

Found: 814.7697 (M+3H).

Preparation of Example 13146

Example 13146 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-[p-Fluorophenylalanine]-Pip-Asp-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-Glu(OtBu)-[Modified resin 13A].After deprotection following Global Deprotection procedure B andcyclization according to Cyclization method B, the compound was purifiedas follows: The residue was split and two injections were made to a30×150 XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over25 min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1%TFA; B is 90% ACN/10% water/0.1% TFA). ACN was removed from thefractions on the rotovap, and the aqueous portion was lyophilized.Analytical data supported the submission of(6S,14S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-31-((1H-indol-3-yl)methyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-50-(carboxymethyl)-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-7-(4-fluorobenzyl)-35-hydroxy-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,9,16-tetraoxo-2,5,10,15-tetraazadotriacontane-6,14,32-tricarboxylicacid, 2 TFA (4.03 mg, 1.441 μmol). Analysis condition 13A: rt 11.50 min.Analysis condition 13B: rt 9.55 min.; ESI-HRMS(+) m/z: Calculated:1228.6529 (M+2H) Found: 1228.6581 (M+2H).

Preparation of Example 13147

Example 13147 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-[p-Fluorophenylalanine]-Pip-Asp-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-Asp(OtBu)-[Modified resin 13B].After deprotection following Global Deprotection procedure B andcyclization according to Cyclization method B, the compound was purifiedas follows: The residue was split and two injections were made to a30×150 XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over25 min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1%TFA; B is 90% ACN/10% water/0.1% TFA). ACN was removed from thefractions on the rotovap, and the aqueous portion was lyophilized.Analytical data supported the submission of(6S,12S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-3141H-indol-3-yl)methyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-50-(carboxymethyl)-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-7-(4-fluorobenzyl)-35-hydroxy-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,8,14-tetraoxo-2,5,9,13-tetraazatriacontane-6,12,30-tricarboxylicacid, 2 TFA (17.72 mg, 6.60 μmol). Analysis condition 13A: rt 11.38 min.Analysis condition 13B: rt 9.47 min.; ESI-HRMS(+) m/z: Calculated:1214.6355 (M+2H)

Found: 1214.6373 (M+2H).

Preparation of Example 13148

Example 13148 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-[p-Fluorophenylalanine]-Pip-Asp-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-Glu(OtBu)-[Modified resin 13B].After deprotection following Global Deprotection procedure B andcyclization according to Cyclization method B, the compound was purifiedas follows: The residue was split and multiple injections were made to a30×150 XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over25 min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1%TFA; B is 90% ACN/10% water/0.1% TFA). ACN was removed from thefractions on the rotovap, and the aqueous portion was lyophilized.Analytical data supported the submission of(6S,13S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-31-((1H-indol-3-yl)methyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-50-(carboxymethyl)-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-7-(4-fluorobenzyl)-35-hydroxy-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,9,15-tetraoxo-2,5,10,14-tetraazahentriacontane-6,13,31-tricarboxylicacid, 2 TFA (13.42 mg, 4.77 μmol). Analysis condition 13A: rt 12.60 min.Analysis condition 13B: rt 10.67 min.; ESI-HRMS(+) m/z: Calculated:1221.6451 (M+2H) Found: 1221.6421 (M+2H).

Preparation of Example 13149

Example 13149 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[Modified resin 13C]. Afterdeprotection following Global Deprotection procedure B and cyclizationaccording to Cyclization method B, the compound was purified as follows:The residue was split and multiple injections were made to a 30×150XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over 25min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1% TFA; Bis 90% ACN/10% water/0.1% TFA). ACN was removed from the fractions onthe rotovap, and the aqueous portion was lyophilized. Analytical datasupported the submission of(6S,13S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-31-((1H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-25-((1-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,10,15-tetraoxo-2,5,9,14-tetraazahentriacontane-6,13,31-tricarboxylicacid, 2 TFA (22.17 mg, 7.64 μmol). Analysis condition 13A: rt 11.04 min.Analysis condition 13B: rt 9.76 min.; ESI-HRMS(+) m/z: Calculated:1220.1553 (M+2H)

Found: 1220.1526 (M+2H).

Preparation of Example 13150

Example 13150 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-[p-Fluorophenylalanine]-Pip-Asp-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[Modified resin 13C]. Afterdeprotection following Global Deprotection procedure B and cyclizationaccording to Cyclization method B, the compound was purified as follows:The residue was split and multiple injections were made to a 30×150XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over 25min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1% TFA; Bis 90% ACN/10% water/0.1% TFA). ACN was removed from the fractions onthe rotovap, and the aqueous portion was lyophilized. Analytical datasupported the submission of(6S,13S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-31-((1H-indol-3-yl)methyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-50-(carboxymethyl)-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-7-(4-fluorobenzyl)-35-hydroxy-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,10,15-tetraoxo-2,5,9,14-tetraazahentriacontane-6,13,31-tricarboxylicacid, 2 TFA (12.96 mg, 4.37 μmol). Analysis condition 13A: rt 12.52 min.Analysis condition 13B: rt 10.61 min.; ESI-HRMS(+) m/z: Calculated:1221.6451 (M+2H)

Found: 1221.6429 (M+2H).

Preparation of Example 13151

Example 13151 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-[p-Methoxyphenylalanine]-Pip-Asn-Pro-His-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[Modified resin 13C]. Afterdeprotection following Global Deprotection procedure B and cyclizationaccording to Cyclization method B, the compound was purified as follows:The residue was split and multiple injections were made to a 30×150XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over 25min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1% TFA; Bis 90% ACN/10% water/0.1% TFA). ACN was removed from the fractions onthe rotovap, and the aqueous portion was lyophilized. Analytical datasupported the submission of(6S,13S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-42-((1H-imidazol-4-yl)methyl)-31-((1H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-19,22-dibutyl-25-((1-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-16,39-diisobutyl-7-(4-methoxybenzyl)-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,10,15-tetraoxo-2,5,9,14-tetraazahentriacontane-6,13,31-tricarboxylicacid, 2 TFA (18.49 mg, 6.63 μmol). Analysis condition 13A: rt 11.80 min.Analysis condition 13B: rt 10.12 min.; ESI-HRMS(+) m/z: Calculated:1252.6685 (M+2H)

Found: 1252.6670 (M+2H).

Preparation of Example 13152

Example 13152 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[Modified resin 13D]. Afterdeprotection following Global Deprotection procedure B and cyclizationaccording to Cyclization method B, the compound was purified as follows:The residue was split and multiple injections were made to a 30×150XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over 25min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1% TFA; Bis 90% ACN/10% water/0.1% TFA). ACN was removed from the fractions onthe rotovap, and the aqueous portion was lyophilized. Analytical dataconfirms(6S,12S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-3141H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,9,14-tetraoxo-2,5,8,13-tetraazatriacontane-6,12,30-tricarboxylicacid, 2 TFA (20.36 mg, 6.90 μmol, 6.90% yield). Analysis condition 13A:rt 10.51 min. Analysis condition 13B: rt 9.22 min.; ESI-HRMS(+) m/z:Calculated: 1213.1474 (M+2H) Found: 1213.1440 (M+2H).

Preparation of Example 13153

Example 13153 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-[N-Me]Ala-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[Modified resin 13D]. Afterdeprotection following Global Deprotection procedure B and cyclizationaccording to Cyclization method B, the compound was purified as follows:The residue was split and multiple injections were made to a 30×150XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over 25min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1% TFA; Bis 90% ACN/10% water/0.1% TFA). ACN was removed from the fractions onthe rotovap, and the aqueous portion was lyophilized. Analytical datasupported the submission of(6S,12S)-1-((6S,9S,12S,18R,21S,24S,27S,30S,33S,36S,38aS,40R,44S,47S,49aS)-36-((1H-indol-3-yl)methyl)-6-(2-amino-2-oxoethyl)-33-(2-aminoethyl)-47-(aminomethyl)-24,27-dibutyl-30-((1-(carboxymethyl)-1H-indol-3-yl)methyl)-40-hydroxy-12-(4-hydroxybenzyl)-21,44-diisobutyl-9,10,25,28-tetramethyl-5,8,11,14,20,23,26,29,32,35,38,43,46,49-tetradecaoxooctatetracontahydrodipyrrolo[2,1-g<sub>1</sub>:2′,1′-x][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-18-yl)-1,4,9,14-tetraoxo-2,5,8,13-tetraazatriacontane-6,12,30-tricarboxylicacid, 2 TFA (21.88 mg, 7.91 μmol, 7.91% yield). Analysis condition 13A:rt 10.37 min. Analysis condition 13B: rt 9.04 min.; ESI-HRMS(+) m/z:Calculated: 1200.1396 (M+2H) Found: 1200.1373 (M+2H).

Preparation of Example 13154

Example 13154 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-[p-Methoxyphenylalanine]-Pip-Asn-Pro-His-Gln-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[Modified resin 13D]. Afterdeprotection following Global Deprotection procedure B and cyclizationaccording to Cyclization method B, the compound was purified as follows:The residue was split and multiple injections were made to a 30×150XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over 25min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1% TFA; Bis 90% ACN/10% water/0.1% TFA). ACN was removed from the fractions onthe rotovap, and the aqueous portion was lyophilized. Analytical datasupported the submission of(6S,12S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-42-((1H-imidazol-4-yl)methyl)-31-((1H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-39-(3-amino-3-oxopropyl)-28-(2-aminoethyl)-19,22-dibutyl-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-16-isobutyl-7-(4-methoxybenzyl)-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,9,14-tetraoxo-2,5,8,13-tetraazatriacontane-6,12,30-tricarboxylicacid, 2 TFA (6.71 mg, 2.209 μmol, 2.209% yield). Analysis condition 13A:rt 11.04 min. Analysis condition 13B: rt 9.34 min.; ESI-HRMS(+) m/z:Calculated: 1253.1480 (M+2H) Found: 1253.1443 (M+2H).

Preparation of Example 13155

Example 13155 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-[p-Methoxyphenylalanine]-Pip-Asn-Pro-His-Gln-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[Modified resin 13D]. Afterdeprotection following Global Deprotection procedure B and cyclizationaccording to Cyclization method B, the compound was purified as follows:The residue was split and multiple injections were made to a 30×150XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over 25min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1% TFA; Bis 90% ACN/10% water/0.1% TFA). ACN was removed from the fractions onthe rotovap, and the aqueous portion was lyophilized. Analytical datasupported the submission of(6S,12S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-42-((1H-imidazol-4-yl)methyl)-31-((1H-indol-3-yl)methyl)-39-(3-amino-3-oxopropyl)-28-(2-aminoethyl)-19,22-dibutyl-50-(carboxymethyl)-25-((1-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-16-isobutyl-7-(4-methoxybenzyl)-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,9,14-tetraoxo-2,5,8,13-tetraazatriacontane-6,12,30-tricarboxylicacid, 2 TFA (10.61 mg, 3.49 μmol, 3.49% yield). Analysis condition 13A:rt 11.19 min. Analysis condition 13B: rt 9.48 min.; ESI-HRMS(+) m/z:Calculated: 1253.6400 (M+2H) Found: 1253.6372 (M+2H).

Preparation of Example 13156

Example 13156 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-[p-Methoxyphenylalanine]-Pip-Asn-Pro-His-Gln-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-Asp(OtBu)-[Modified resin 13B].After deprotection following Global Deprotection procedure B andcyclization according to Cyclization method B, the compound was purifiedas follows: The residue was split and multiple injections were made to a30×150 XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over25 min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1%TFA; B is 90% ACN/10% water/0.1% TFA). ACN was removed from thefractions on the rotovap, and the aqueous portion was lyophilized.Analytical data supported the submission of(6S,12S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-42-((1H-imidazol-4-yl)methyl)-31-((1H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-39-(3-amino-3-oxopropyl)-28-(2-aminoethyl)-19,22-dibutyl-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-16-isobutyl-7-(4-methoxybenzyl)-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,8,14-tetraoxo-2,5,9,13-tetraazatriacontane-6,12,30-tricarboxylicacid, 2 TFA (10.73 mg, 3.53 μmol, 3.53% yield). Analysis condition 13A:rt 10.92 min. Analysis condition 13B: rt 9.27 min.; ESI-HRMS(+) m/z:Calculated: 1253.1480 (M+2H) Found: 1253.1448 (M+2H).

Preparation of Example 13157

Example 13157 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-[p-Methoxyphenylalanine]-Pip-Asp-Pro-His-Gln-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-Asp(OtBu)-[Modified resin 13B].After deprotection following Global Deprotection procedure B andcyclization according to Cyclization method B, the compound was purifiedas follows: The residue was split and multiple injections were made to a30×150 XSelect CSH Prep C18 5 um OBD column, 40 mL/min, 20 to 65% B over25 min, 5 min at 100% B, 5 min at 20% B (A is 90% water/10% ACN/0.1%TFA; B is 90% ACN/10% water/0.1% TFA). ACN was removed from thefractions on the rotovap, and the aqueous portion was lyophilized.Analytical data supported the submission of(6S,12S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-42-((1H-imidazol-4-yl)methyl)-31-((1H-indol-3-yl)methyl)-39-(3-amino-3-oxopropyl)-28-(2-aminoethyl)-19,22-dibutyl-50-(carboxymethyl)-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-16-isobutyl-7-(4-methoxybenzyl)-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,8,14-tetraoxo-2,5,9,13-tetraazatriacontane-6,12,30-tricarboxylicacid, 2 TFA (12.14 mg, 4.22 μmol, 4.22% yield). Analysis condition 13A:rt 11.09 min. Analysis condition 13B: rt 9.42 min.; ESI-HRMS(+) m/z:Calculated: 1253.6400 (M+2H) Found: 1253.6367 (M+2H).

Preparation of Example 13158

Example 13158 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[morpholine-3R-carboxylicacid]-Asp(OtBu)-[Modified resin 13B]. After deprotection followingGlobal Deprotection procedure B and cyclization according to Cyclizationmethod B, the compound was purified as follows: The residue was splitand multiple injections were made to a 30×150 XSelect CSH Prep C18 5 umOBD column, 40 mL/min, 20 to 65% B over 25 min, 5 min at 100% B, 5 minat 20% B (A is 90% water/10% ACN/0.1% TFA; B is 90% ACN/10% water/0.1%TFA). ACN was removed from the fractions on the rotovap, and the aqueousportion was lyophilized. Analytical data supported the submission of18-(((S)-3-((R)-4-((S)-3-(2-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-3141H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-25-((1-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontine-13-carboxamido)acetamido)-3-carboxypropanoyl)morpholine-3-carboxamido)-1-carboxypropyl)amino)-18-oxooctadecanoicacid, 2 TFA (22.68 mg, 7.38 μmol, 7.38% yield). Analysis condition 13A:rt 10.15 min. Analysis condition 13B: rt 8.82 min.; ESI-HRMS(+) m/z:Calculated: 1269.6713 (M+2H) Found: 1269.6671 (M+2H).

Preparation of Example 13159

Example 13159 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[2-(2-aminoethoxy)aceticacid]-Asp(OtBu)-[Modified resin 13B]. After deprotection followingGlobal Deprotection procedure B and cyclization according to Cyclizationmethod B, the compound was purified as follows: The residue was splitand multiple injections were made to a 30×150 XSelect CSH Prep C18 5 umOBD column, 40 mL/min, 20 to 65% B over 25 min, 5 min at 100% B, 5 minat 20% B (A is 90% water/10% ACN/0.1% TFA; B is 90% ACN/10% water/0.1%TFA). ACN was removed from the fractions on the rotovap, and the aqueousportion was lyophilized. Analytical data supported the submission of(6S,18S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-3141H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-25-((1-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,8,14,20-pentaoxo-12-oxa-2,5,9,15,19-pentaazahexatriacontane-6,18,36-tricarboxylicacid, 2 TFA (12.14 mg, 3.97 μmol, 3.97% yield). Analysis condition 13A:rt 10.01 min. Analysis condition 13B: rt 8.70 min.; ESI-HRMS(+) m/z:Calculated: 1263.6713 (M+2H) Found: 1263.6668 (M+2H).

Preparation of Example 13160

Example 13160 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[3-aminopropanoicacid]-Asp(OtBu)-[Modified resin 13B]. After deprotection followingGlobal Deprotection procedure B and cyclization according to Cyclizationmethod B, the compound was purified as follows: The residue was splitand multiple injections were made to a 30×150 XSelect CSH Prep C18 5 umOBD column, 40 mL/min, 20 to 65% B over 25 min, 5 min at 100% B, 5 minat 20% B (A is 90% water/10% ACN/0.1% TFA; B is 90% ACN/10% water/0.1%TFA). ACN was removed from the fractions on the rotovap, and the aqueousportion was lyophilized. Analytical data supported the submission of(6S,16S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-3141H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,8,12,18-pentaoxo-2,5,9,13,17-pentaazatetratriacontane-6,16,34-tricarboxylicacid, 2 TFA (11.56 mg, 4.03 μmol). Analysis condition 13A: rt 10.00 min.Analysis condition 13B: rt 8.69 min.; ESI-HRMS(+) m/z: Calculated:1248.6660 (M+2H) Found: 1248.6622 (M+2H).

Preparation of Example 13161

Example 13161 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[3-(2-aminoethoxy)propanoicacid]-Asp(OtBu)-[Modified resin 13B]. After deprotection followingGlobal Deprotection procedure B and cyclization according to Cyclizationmethod B, the compound was purified as follows: The residue was splitand multiple injections were made to a 30×150 XSelect CSH Prep C18 5 umOBD column, 40 mL/min, 20 to 65% B over 25 min, 5 min at 100% B, 5 minat 20% B (A is 90% water/10% ACN/0.1% TFA; B is 90% ACN/10% water/0.1%TFA). ACN was removed from the fractions on the rotovap, and the aqueousportion was lyophilized. Analytical data supported the submission of(6S,19S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-3141H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-25-((1-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,8,15,21-pentaoxo-12-oxa-2,5,9,16,20-pentaazaheptatriacontane-6,19,37-tricarboxylicacid, 2 TFA (22.02 mg, 7.55 μmol, 7.55% yield). Analysis condition 13A:rt 10.01 min. Analysis condition 13B: rt 8.69 min.; ESI-HRMS(+) m/z:Calculated: 1270.6791 (M+2H) Found: 1270.6751 (M+2H).

Preparation of Example 13162

Example 13162 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[4-aminobutanoicacid]-Asp(OtBu)-[Modified resin 13B]. After deprotection followingGlobal Deprotection procedure B and cyclization according to Cyclizationmethod B, the compound was purified as follows: The residue was splitand multiple injections were made to a 30×150 XSelect CSH Prep C18 5 umOBD column, 40 mL/min, 20 to 65% B over 25 min, 5 min at 100% B, 5 minat 20% B (A is 90% water/10% ACN/0.1% TFA; B is 90% ACN/10% water/0.1%TFA). ACN was removed from the fractions on the rotovap, and the aqueousportion was lyophilized. Analytical data supported the submission of(6S,17S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-3141H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-2541-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,8,13,19-pentaoxo-2,5,9,14,18-pentaazapentatriacontane-6,17,35-tricarboxylicacid, 2 TFA (17.91 mg, 6.08 μmol, 6.08% yield). Analysis condition 13A:rt 10.07 min. Analysis condition 13B: rt 8.73 min.; ESI-HRMS(+) m/z:Calculated: 1255.6738 (M+2H) Found: 1255.6692 (M+2H).

Preparation of Example 13163

Example 13163 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[3-(2-(2-aminoethoxy)ethoxy)propanoicacid]-Asp(OtBu)-[Modified resin 13B]. After deprotection followingGlobal Deprotection procedure B and cyclization according to Cyclizationmethod B, the compound was purified as follows: The residue was splitand multiple injections were made to a 30×150 XSelect CSH Prep C18 5 umOBD column, 40 mL/min, 20 to 65% B over 25 min, 5 min at 100% B, 5 minat 20% B (A is 90% water/10% ACN/0.1% TFA; B is 90% ACN/10% water/0.1%TFA). ACN was removed from the fractions on the rotovap, and the aqueousportion was lyophilized. Analytical data supported the submission of(6S,22S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-31-((1H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-25-((1-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,8,18,24-pentaoxo-12,15-dioxa-2,5,9,19,23-pentaazatetracontane-6,22,40-tricarboxylicacid, 2 TFA (18.29 mg, 5.85 μmol, 5.85% yield). Analysis condition 13A:rt 10.00 min. Analysis condition 13B: rt 8.68 min.; ESI-HRMS(+) m/z:Calculated: 1292.6713 (M+2H) Found: 1292.6887 (M+2H).

Preparation of Example 13164

Example 13164 was synthesized on a 0.1 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr-Pip-Asn-Pro-Dap-Leu-Hyp-Trp-Dab-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-[N-Me]Nle-[N-Me]Nle-Leu-Cys-Gly-[5-aminopentanoicacid]-Asp(OtBu)-[Modified resin 13B]. After deprotection followingGlobal Deprotection procedure B and cyclization according to Cyclizationmethod B, the compound was purified as follows: The residue was splitand multiple injections were made to a 30×150 XSelect CSH Prep C18 5 umOBD column, 40 mL/min, 20 to 65% B over 25 min, 5 min at 100% B, 5 minat 20% B (A is 90% water/10% ACN/0.1% TFA; B is 90% ACN/10% water/0.1%TFA). ACN was removed from the fractions on the rotovap, and the aqueousportion was lyophilized. Analytical data supported the submission of(6S,18S)-1-((7S,13R,16S,19S,22S,25S,28S,31S,33aS,35R,39S,42S,44aS,50S,52aR)-31-((1H-indol-3-yl)methyl)-50-(2-amino-2-oxoethyl)-28-(2-aminoethyl)-42-(aminomethyl)-19,22-dibutyl-25-((1-(carboxymethyl)-1H-indol-3-yl)methyl)-35-hydroxy-7-(4-hydroxybenzyl)-16,39-diisobutyl-20,23-dimethyl-6,9,15,18,21,24,27,30,33,38,41,44,49,52-tetradecaoxopentacontahydro-1H-pyrido[1,2-g]dipyrrolo[1,2-m:1′,2′-v][1,4,7,10,13,16,19,22,25,28,31,34,37,40,43]thiatetradecaazacyclopentatetracontin-13-yl)-1,4,8,14,20-pentaoxo-2,5,9,15,19-pentaazahexatriacontane-6,18,36-tricarboxylicacid, 2 TFA (10.63 mg, 3.48 μmol, 3.48% yield). Analysis condition 13A:rt 10.06 min. Analysis condition 13B: rt 8.73 min.; ESI-HRMS(+) m/z:Calculated: 1262.6816 (M+2H) Found: 1262.6773 (M+2H).

Preparation of INT-1400A

The following peptide was synthesized on a 0.2 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-mAla-Asp-Pro-His-Lys-Hyp-Trp-Lys-Trp-mNle-mNle-Glu-Cys-Gly-[(S)-propargylglycine]-Gly.After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 15-55% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 97.7 mg, and its estimated purity by LCMSanalysis was 99%. Analysis condition A: Retention time=1.39 min;ESI-MS(+) m/z 1046.9 (M+2H), most abundant ion; Analysis condition B:Retention time=2.05 min; ESI-MS(+) m/z 1046.8 (M+2H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 1046.5091 (M+2H) Found: 1046.5058 (M+2H).

Preparation of INT-1400B

The following peptide was synthesized on a 0.2 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-mAla-Asp-Pro-His-Lys-Hyp-Trp-Lys-Trp-mNle-mNle-Glu-Cys-Gly-[(S)-propargylglycine]-Asp;After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 15-55% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 59.5 mg, and its estimated purity by LCMSanalysis was 100%. Analysis condition A: Retention time=1.69 min;ESI-MS(+) m/z 1076.2 (M+2H), most abundant ion; Analysis condition B:Retention time=2.91 min; ESI-MS(+) m/z 1076.1 (M+2H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 1075.5118 (M+2H) Found: 1075.5094 (M+2H).

Preparation of INT-1400C

The following peptide was synthesized on a 0.1 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-mAla-Asp-Pro-His-Gln-Hyp-Trp-Ser-Trp-mNle-mNle-Glu-Cys-Gly-[(S)-propargylglycine]-Gly.After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation.

The yield of the product was 20.2 mg, and its estimated purity by LCMSanalysis was 98%. Analysis condition A: Retention time=1.40 min;ESI-MS(+) m/z 1026.5 (M+2H), most abundant ion; Analysis condition B:Retention time=2.79 min; ESI-MS(+) m/z 1026.5 (M+2H), most abundant ion;ESI-HRMS(+) m/z:

Calculated: 1025.9594 (M+2H) Found: 1025.9591 (M+2H).

Preparation of INT-1400D

The following peptide was synthesized on a 0.2 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-mAla-Asp-Pro-His-Lys-Hyp-Trp-Lys-“Trp”-mNle-mNle-Glu-Cys-Gly-[(S)-propargylglycine]-Gly.After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 35-75% B over 30 minutes, then a 6-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 57.9 mgs, and its estimated purity by LCMS analysiswas 100%. Analysis condition A: Retention time=1.16 min; ESI-MS(−) m/z1076.6 (M+2H), most abundant ion; Analysis condition B: Retentiontime=2.23 min; ESI-MS(+) m/z 1076.3 (M+2H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 1075.5118 (M+2H) Found: 1075.5086 (M+2H).

Preparation of INT-1400E

The following peptide was synthesized on a 0.2 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-mAla-Asp-Pro-His-Lys-Hyp-Trp-Lys-“Trp”-mNle-mNle-Glu-Cys-Gly-[(S)-propargylglycine]-Asp.After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 30-75% B over 30 minutes, then a 6-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 66.6 mgs, and its estimated purity by LCMS analysiswas 94%. Analysis condition A: Retention time=1.12 min; ESI-MS(−) m/z1105.5 (M+2H), most abundant ion; Analysis condition B: Retentiontime=2.19 min; ESI-MS(+) m/z 11055.4 (M+2H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 1104.5146 (M+2H). Found: 1104.5115 (M+2H).

Preparation of INT-1400F

The following peptide was synthesized on a 0.2 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-mAla-Asp-Pro-His-Lys-Hyp-Trp-Lys-Trp-mNle-mNle-Glu-Cys-Gly-[(S)-propargylglycine].After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 35-75% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 87.3 mg, and its estimated purity by LCMS analysiswas 99%. Analysis condition A: Retention time=1.39 min; ESI-MS(+) m/z1019.0 (M+2H), most abundant ion; Analysis condition B: Retentiontime=2.53 min; ESI-MS(+) m/z 1018.9 (M+2H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 1017.9984 (M+2H) Found: 1017.9944 (M+2H).

Preparation of INT-1400G

The following peptide was synthesized on a 0.2 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-mAla-Asn-Pro-Dap-Leu-Hyp-Trp-Ser-Trp-mNle-mNle-Arg-Cys-Gly-[(S)-propargylglycine].After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 35-80% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 44.2 mg, and its estimated purity by LCMS analysiswas 98%. Analysis condition A: Retention time=1.62 min; ESI-MS(+) m/z977.7 (M+2H), most abundant ion; Analysis condition B: Retentiontime=3.16 min; ESI-MS(+) m/z 977.7 (M+2H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 977.4933 (M+2H) Found: 977.4906 (M+2H).

Preparation of INT-1400H

The following peptide was synthesized on a 0.2 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-mAla-Asp-Pro-His-Gln-Hyp-Trp-Ser-Trp-mNle-mNle-Glu-Cys-Gly-[(S)-propargylglycine];After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 5-45% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 23.9 mg, and its estimated purity by LCMSanalysis was 98%. Analysis condition A: Retention time=1.45 min;ESI-MS(+) m/z 997.5 (M+2H), most abundant ion; Analysis condition B:Retention time=2.87 min; ESI-MS(+) m/z 997.9 (M+2H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 997.4487 (M+2H) Found: 997.4486 (M+2H).

Preparation of INT-1400I

The following peptide was synthesized on a 0.1 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-mAla-Asn-Pro-Dap-Leu-Hyp-Trp-Ser-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-mNle-mNle-E-Cys-Gly-[(S)-propargylglyeine]. After deprotection andcyclization according to the procedures above, the compound was purifiedas follows: The crude material was purified via preparative LC/MS withthe following conditions: Column: XBridge C18, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 20-40% B over 20 minutes, then a 5-minute hold at100% B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 42.1 mg, and its estimated purity by LCMS analysis was 98%. Analysiscondition A: Retention time=1.26 min; ESI-MS(+) m/z 993.3 (M+2H), mostabundant ion; Analysis condition B: Retention time=2.68 min; ESI-MS(+)m/z 993.3 (M+2H), most abundant ion; ESI-HRMS(+) m/z: Calculated:992.9667 (M+2H) Found: 992.9660 (M+2H).

Preparation of INT-1400J

The following peptide was synthesized on a 0.2 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr-mAla-Asp-Pro-His-Lys-Hyp-Trp-Lys-[(S)-2-amino-3-(1-(carboxymethyl)-1H-indol-3-yl)propanoicacid]-mNle-mNle-Glu-Cys-Gly-[(S)-propargylglycine]; After deprotectionand cyclization according to the procedures above, the compound waspurified as follows: The crude material was purified via preparativeLC/MS with the following conditions: Column: XBridge C18, 19×200 mm,5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10-mMammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mMammonium acetate; Gradient: 0-40% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 78.3 mg, and its estimated purity by LCMS analysiswas 100%. Analysis condition A: Retention time=1.25 min; ESI-MS(+) m/z1047.6 (M+2H), most abundant ion

Analysis condition B: Retention time=2.65 min; ESI-MS(+) m/z 1047.4(M+2H), most abundant ion ESI-HRMS(+) m/z: Calculated: 1047.0011 (M+2H)

Found: 1046.9960 (M+2H).

Preparation of INT-1400K

The following peptide was synthesized on a 0.1 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-F3Phe-mPhe-mNle-dPro-Asp-Val-mPhe-Orn-dPro-Trp-Tyr-Leu-Cys-Gly-[(S)-propargylglycine].After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 25-65% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation.

The yield of the product was 88.5 mg, and its estimated purity by LCMSanalysis was 95%. Analysis condition A: Retention time=1.87 min;ESI-MS(+) m/z 975.7 (M+2H), most abundant ion. Analysis condition B:Retention time=3.25 min; ESI-MS(+) m/z 975.1 (M+2H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 974.4593 (M+2H)

Found: 974.4571 (M+2H).

Preparation of INT-1400L

The following peptide was synthesized on a 0.1 mmol scale according tothe procedures above. The underlined steps employed the double-couplingprocedure.ClAc-F3Phe-mPhe-mNle-dHyp-Asp-Val-mPhe-Asp-dPro-Trp-Tyr-Leu-Cys-Gly-[(S)-propargylglycine];After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 50-90% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 70.7 mg, and its estimated purity by LCMS analysiswas 100%. Analysis condition A: Retention time=1.47 min; ESI-MS(+) m/z983.5 (M+2H), most abundant ion; Analysis condition B: Retentiontime=2.74 min; ESI-MS(+) m/z 983.7 (M+2H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 982.9306 (M+2H) Found: 982.9300 (M+2H).

Analytical Data:

Mass Spectrometry: “ESI-MS(+)” signifies electrospray ionization massspectrometry performed in positive ion mode; “ESI-MS(−)” signifieselectrospray ionization mass spectrometry performed in negative ionmode; “ESI-HRMS(+)” signifies high-resolution electrospray ionizationmass spectrometry performed in positive ion mode; “ESI-HRMS(−)”signifies high-resolution electrospray ionization mass spectrometryperformed in negative ion mode. The detected masses are reportedfollowing the “m/z” unit designation. Compounds with exact massesgreater than 1000 were often detected as double-charged ortriple-charged ions.

Analysis Condition A:

Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50°C.; Gradient: 0% B, 0-100% B over 3 minutes, then a 0.5-minute hold at100% B; Flow: 1 mL/min; Detection: UV at 220 nm.

Analysis Condition B:

Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 methanol:water with 10 mM ammonium acetate; Mobile Phase B: 95:5methanol:water with 10 mM ammonium acetate; Temperature: 50° C.;Gradient: 0% B, 0-100% B over 3 minutes, then a 0.5-minute hold at 100%B; Flow: 0.5 mL/min; Detection: UV at 220 nm.

Analysis Condition C:

Column: Waters Aquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; MobilePhase A: 100% Water:0.05% TFA; Mobile Phase B: 100% Acetonitrile:0.05%TFA; Temperature: 40° C.; Gradient: 2-98% B over 1.5 minutes, then a0.5-minute hold at 100% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

Analysis Condition D:

Column: PHENOMENEX-LUNA 2.0×30 mm 3 um; Mobile Phase A: 90% Water—10%Methanol—0.1% TFA; Mobile Phase B: 10% Water—90% Methanol—0.1% TFA;Gradient: 0-100% B over 2 minutes, then a 1 to 4 minute hold at 100% B;Flow: 1 mL/min; Detection: UV at 220 nm.

Analysis Condition E:

Column: Xbridge Phenyl, 3.0×150 mm, 3.5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10 mM ammonium acetate; Gradient: 5-100% Bover 15 minutes; Flow: 0.5 mL/min; Detection: UV at 220 nm.

Analysis Condition F:

Column: XBridge C18, 3.0×150 mm, 3.5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10 mM ammonium acetate; Gradient: 10-100% B over30 minutes; Flow: 0.5 mL/min; Detection: UV at 220 nm.

Analysis Condition G:

Column: Waters CSH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A:5:95 acetonitrile:water with 0.1% TFA; Mobile Phase B: 95:5acetonitrile:water with 0.1% TFA; Temperature: 50° C.; Gradient: 0% B,0-100% B over 3 minutes, then a 0.5-minute hold at 100% B; Flow: 1mL/min; Detection: UV at 220 nm.

Analysis Condition H:

Column: Xbridge C18, 3.0×150 mm, 3.5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10 mM ammonium acetate; Gradient: 10-100% B over18 minutes; Flow: 0.5 mL/min; Detection: UV at 220 nm.

Analysis Condition I:

Column: XSelectCSH C18, 3.0×150 mm, 3.5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 0.1% TFA; Mobile Phase B: 95:5acetonitrile:water with 0.1% TFA; Gradient: 10-100% B over 15 minutes;Flow: 1.0 mL/min; Detection: UV at 220 nm.

Analysis Condition J:

Column: Zorbax Bonus RP, 3.0×150 mm, 3.5-μm particles; Mobile Phase A:5:95 acetonitrile:water with 0.1% TFA; Mobile Phase B: 95:5acetonitrile:water with 0.1% TFA; Gradient: 10-100% B over 15 minutes;Flow: 1.0 mL/min; Detection: UV at 220 nm.

Analysis Condition K:

Column: Waters Aquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; MobilePhase A: 100% Water:0.05% TFA; Mobile Phase B: 100% Acetonitrile:0.05%TFA; Temperature: 50° C.; Gradient: 2-98% B over 3.0 minutes, then a0.5-minute hold at 100% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

Preparation of 14051

Intermediate 1400A (20 mg, 9.56 μmol) and(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate (18.38 mg, 0.029 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via Prep-HPLC (SolventA=10% MeOH—90% H₂O—0.1% TFA, Solvent B=90% MeOH—10% H2O—0.1% TFA.Column: PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40 ml/min, 55-100% B,25 min, then a 6-minute hold at 100% B). Fractions containing thedesired product were combined and dried via speed vacuum evaporation.The yield of the product was 7.22 mg, and its estimated purity by LCMSanalysis was 90%. Analysis condition C: Retention time=1.64 min;ESI-MS(+) m/z 1367.8 (M+2H), most abundant ion. Analysis condition D:Retention time=2.48 min; ESI-MS(+) m/z 1367.4 (M+2H), most abundant ion.

Preparation of 14052

Intermediate 1400A (20 mg, 9.56 μmol) and23-azido-3,6,9,12,15,18,21-heptaoxatricosyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate (26.0 mg, 0.029 mmol) were reacted as in the general triazoleformation procedure above to afford crude product. The crude materialwas purified via Prep-HPLC (Solvent A=10% MeOH—90% H₂O—0.1% TFA, SolventB=90% MeOH—10% H2O—0.1% TFA. Column: PHENOMENEX LUNA 30×100 mm, S10,Flow rate: 40 ml/min, 55-100% B, 25 min, then a 10-minute hold at 100%B). Fractions containing the desired product were combined and dried viaspeed vacuum evaporation. The yield of the product was 11 mg, and itsestimated purity by LCMS analysis was 90%. Analysis condition C:Retention time=1.66 min; ESI-MS(+) m/z 1501.5 (M+2H), most abundant ion;Analysis condition D: Retention time=2.63 min; ESI-MS(+) m/z 1001.1(M+3H), most abundant ion.

Preparation of 14053

Intermediate 1400A (20 mg, 9.56 μmol) and17-azido-3,6,9,12,15-pentaoxaheptadecyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate (23.52 mg, 0.029 mmol) were reacted as in the general triazoleformation procedure above to afford crude product. The crude materialwas purified via Prep-HPLC (Solvent A=10% MeOH—90% H2O—0.1% TFA, SolventB=90% MeOH—10% H₂O—0.1% TFA. Column: PHENOMENEX LUNA 30×100 mm, S10,Flow rate: 40 ml/min, 55-100% B, 25 min, then a 7-minute hold at 100%B). Fractions containing the desired product were combined and dried viaspeed vacuum evaporation. The yield of the product was 5.3 mg, and itsestimated purity by LCMS analysis was 90%. Analysis condition C:Retention time=1.66 min; ESI-MS(+) m/z 1457.4 (M+2H), most abundant ion;Analysis condition D: Retention time=3.22 min; ESI-MS(+) m/z 971.8(M+3H), most abundant ion.

Preparation of 14054

Intermediate 1400C (20 mg, 9.75 μmol) and(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate (18.38 mg, 0.029 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via Prep-HPLC (SolventA=10% MeOH—90% H₂O—0.1% TFA, Solvent B=90% MeOH—10% H₂O—0.1% TFA.Column: PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40 ml/min, 55-100% B,25 min, then a 10-minute hold at 100% B). Fractions containing thedesired product were combined and dried via speed vacuum evaporation.The yield of the product was 8.2 mg, and its estimated purity by LCMSanalysis was 90%. Analysis condition C: Retention time=1.76 min;ESI-MS(+) m/z 1347.3 (M+2H), most abundant ion. Analysis condition D:Retention time=2.79 min; ESI-MS(+) m/z 898.3 (M+3H), most abundant ion.

Preparation of 14055

Intermediate 1400C (20 mg, 9.75 μmol) and23-azido-3,6,9,12,15,18,21-heptaoxatricosyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate (26.6 mg, 0.029 mmol) were reacted as in the general triazoleformation procedure above to afford crude product. The crude materialwas purified via Prep-HPLC (Solvent A=10% MeOH—90% H₂O—0.1% TFA, SolventB=90% MeOH—10% H2O—0.1% TFA. Column: PHENOMENEX LUNA 30×100 mm, S10,Flow rate: 40 ml/min, 55-100% B, 25 min, then a 15-minute hold at 100%B). Fractions containing the desired product were combined and dried viaspeed vacuum evaporation. The yield of the product was 9.0 mg, and itsestimated purity by LCMS analysis was 90%. Analysis condition C:Retention time=1.82 min; ESI-MS(+) m/z 1480.9 (M+2H), most abundant ion;Analysis condition D: Retention time=2.99 min; ESI-MS(+) m/z 1480.9(M+2H), most abundant ion.

Preparation of 14056

Intermediate 1400B (20 mg, 9.30 μmol) and(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate (17.88 mg, 0.028 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via Prep-HPLC (SolventA=10% MeOH—90% H2O—0.1% TFA, Solvent B=90% MeOH—10% H2O—0.1% TFA.Column: PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40 ml/min, 55-100% B,25 min, then a 6-minute hold at 100% B). Fractions containing thedesired product were combined and dried via speed vacuum evaporation.The yield of the product was 4.3 mg, and its estimated purity by LCMSanalysis was 90%. Analysis condition D: Retention time=2.42 min;ESI-MS(+) m/z 931.4 (M+3H), most abundant ion.

Preparation of Example 14057

Intermediate 1400B (20 mg, 9.30 μmol) and23-azido-3,6,9,12,15,18,21-heptaoxatricosyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate (25.3 mg, 0.028 mmol) were reacted as in the general triazoleformation procedure above to afford crude product. The crude materialwas purified via Prep-HPLC (Solvent A=10% MeOH—90% H₂O—0.1% TFA, SolventB=90% MeOH—10% H2O—0.1% TFA. Column: PHENOMENEX LUNA 30×100 mm, S10,Flow rate: 40 ml/min, 55-100% B, 25 min, then a 10-minute hold at 100%B). Fractions containing the desired product were combined and dried viaspeed vacuum evaporation

The yield of the product was 4.3 mg, and its estimated purity by LCMSanalysis was 90%. Analysis condition C: Retention time=1.44 min;ESI-MS(+) m/z 929.3 (M+2H); Analysis condition D: Retention time=2.66min; ESI-MS(+) m/z 929.8 (M+2H), most abundant ion.

Preparation of Example 14058

Intermediate 1300A (20 mg, 10.15 μmol) and(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate (19.51 mg, 0.030 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via Prep-HPLC (SolventA=10% MeOH—90% H2O—0.1% TFA, Solvent B=90% MeOH—10% H₂O—0.1% TFA.Column: PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40 ml/min, 55-100% B,25 min, then a 10-minute hold at 100% B). Fractions containing thedesired product were combined and dried via speed vacuum evaporation.The yield of the product was 1.5 mg, and its estimated purity by LCMSanalysis was 90%. Analysis condition D: Retention time=2.77 min;ESI-MS(+) m/z 1307.3 (M+2H).

Preparation of Example 14059

Intermediate 1400A (25 mg, 0.012 mmol) and(S)-4-azido-2-palmitamidobutanoic acid (13.71 mg, 0.036 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via Prep-HPLC (SolventA=10% MeOH—90% H2O—0.1% TFA, Solvent B=90% MeOH—10% H₂O—0.1% TFA.Column: PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40 ml/min, 55-100% B,30 min). Fractions containing the desired product were combined anddried via speed vacuum evaporation. The yield of the product was 1.5 mg,and its estimated purity by LCMS analysis was 90%. Analysis condition D:Retention time=2.32 min; ESI-MS(+) m/z 1238.8 (M+2H).

Preparation of Example 14060

Intermediate 1400A (20 mg, 9.56 μmol) and2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl((R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl)succinate (20.99 mg, 0.029 mmol), were reacted as in the generaltriazole formation procedure above to afford crude product. The crudematerial was purified via Prep-HPLC (Solvent A=10% MeOH—90% H₂O—0.1%TFA, Solvent B=90% MeOH—10% H₂O—0.1% TFA. Column: PHENOMENEX LUNA 30×100mm, S10, Flow rate: 40 ml/min, 55-100% B, 30 min, then a 3-minute holdat 100% B). Fractions containing the desired product were combined anddried via speed vacuum evaporation. The yield of the product was 1.5 mg,and its estimated purity by LCMS analysis was 90%. Analysis condition D:Retention time=2.52 min; ESI-MS(+) m/z 1413.1 (M+2H), most abundant ion.

Preparation of Example 14061

Intermediate 1400B (25 mg, 0.012 mmol) and(S)-4-azido-2-palmitamidobutanoic acid (13.34 mg, 0.035 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via Prep-HPLC (SolventA=10% MeOH—90% H₂O—0.1% TFA, Solvent B=90% MeOH—10% H₂O—0.1% TFA.Column: PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40 ml/min, 55-100% B,30 min). Fractions containing the desired product were combined anddried via speed vacuum evaporation The yield of the product was 9.0 mg,and its estimated purity by LCMS analysis was 90%. Analysis condition D:Retention time=2.31 min; ESI-MS(+) m/z 1267.7 (M+2H), most abundant ion.

Preparation of Example 14062

Intermediate 1400C (25 mg, 0.012 mmol) and(S)-4-azido-2-palmitamidobutanoic acid (13.99 mg, 0.037 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via Prep-HPLC (SolventA=10% MeOH—90% H2O—0.1% TFA, Solvent B=90% MeOH—10% H2O—0.1% TFA.Column: PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40 ml/min, 55-100% B,30 min). Fractions containing the desired product were combined anddried via speed vacuum evaporation. The yield of the product was 10.0mg, and its estimated purity by LCMS analysis was 90%. Analysiscondition D: Retention time=2.47 min; ESI-MS(+) m/z 1217.9 (M+2H), mostabundant ion.

Preparation of Example 14063

Intermediate 1400A (20 mg, 9.56 μmol) and13-azido-2,5,8,11-tetraoxatridecane (6.69 mg, 0.029 mmol) were reactedas in the general triazole formation procedure above to afford crudeproduct. The crude material was purified via Prep-HPLC (Solvent A=10%MeOH—90% H₂O—0.1% TFA, Solvent B=90% MeOH—10% H₂O—0.1% TFA. Column:PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40 ml/min, 35-100% B, 45min). Fractions containing the desired product were combined and driedvia speed vacuum evaporation. The yield of the product was 9.0 mg, andits estimated purity by LCMS analysis was 90%. Analysis condition D:Retention time=1.83 min; ESI-MS(+) m/z 1163.7 (M+2H), most abundant ion.

Preparation of Example 14064

Intermediate 1400A (25 mg, 0.012 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (14.79mg, 0.036 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia Prep-HPLC (Solvent A=10% MeOH—90% H₂O—0.1% TFA, Solvent B=90%MeOH—10% H₂O—0.1% TFA. Column: PHENOMENEX LUNA 30×100 mm, S10, Flowrate: 40 ml/min, 45-100% B, 40 min, then a 5-minute hold at 100% B).Fractions containing the desired product were combined and dried viaspeed vacuum evaporation. The yield of the product was 20.0 mg, and itsestimated purity by LCMS analysis was 90%. Analysis condition D:Retention time=2.09 min; ESI-MS(+) m/z 1253.6 (M+2H).

Preparation of Example 14065

Intermediate 1400B (25 mg, 0.012 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (14.39mg, 0.035 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia Prep-HPLC (Solvent A=10% MeOH—90% H2O—0.1% TFA, Solvent B=90%MeOH—10% H₂O—0.1% TFA. Column: PHENOMENEX LUNA 30×100 mm, S10, Flowrate: 40 ml/min, 50-100% B, 50 min). Fractions containing the desiredproduct were combined and dried via speed vacuum evaporation.

The yield of the product was 13.0 mg, and its estimated purity by LCMSanalysis was 90%. Analysis condition D: Retention time=2.15 min;ESI-MS(+) m/z 1282.5 (M+2H), most abundant ion.

Preparation of Example 14066

Intermediate 1400G (25 mg, 0.013 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (6.33mg, 0.015 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia Prep-HPLC (Solvent A=10% MeOH—90% H₂O—0.1% TFA, Solvent B=90%MeOH—10% H₂O—0.1% TFA. Column: PHENOMENEX LUNA 30×100 mm, S10, Flowrate: 40 ml/min, 50-90% B, 60 min). Fractions containing the desiredproduct were combined and dried via speed vacuum evaporation. The yieldof the product was 10.0 mg, and its estimated purity by LCMS analysiswas 90%. Analysis condition D: Retention time=2.18 min; ESI-MS(+) m/z1184.6 (M+2H), most abundant ion.

Preparation of Example 14067

Intermediate 1400F (25 mg, 0.013 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (7.60mg, 0.018 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia Prep-HPLC (Solvent A=10% MeOH—90% H₂O—0.1% TFA, Solvent B=90%MeOH—10% H₂O—0.1% TFA. Column: PHENOMENEX LUNA 30×100 mm, S10, Flowrate: 40 ml/min, 55-90% B, 60 min). Fractions containing the desiredproduct were combined and dried via speed vacuum evaporation. The yieldof the product was 2.0 mg, and its estimated purity by LCMS analysis was90%. Analysis condition D: Retention time=2.16 min; ESI-MS(+) m/z 1225.1(M+2H), most abundant ion.

Preparation of Example 14068

Intermediate 1400H (25 mg, 0.013 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (6.21mg, 0.015 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeShield RP18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 20-60% B over30 minutes, then a 15-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 6.5 mg, and its estimatedpurity by LCMS analysis was 100%. Analysis condition B: Retentiontime=1.76 min; ESI-MS(+) m/z 1204.3 (M+2H), most abundant ion.ESI-HRMS(+) m/z: Calculated: 1203.5830 (M+2H)

Found: 1203.5818 (M+2H).

Preparation of Example 14069

Intermediate 1400C (19.5 mg, 9.51 μmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (4.71mg, 0.011 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeShield RP18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-55% B over30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 5.2 mg, and its estimatedpurity by LCMS analysis was 96%. Analysis condition B: Retentiontime=1.75 min; ESI-MS(+) m/z 1232.8 (M+2H), most abundant ion.ESI-HRMS(+) m/z: Calculated: 1232.0937 (M+2H)

Found: 1232.0902 (M+2H).

Preparation of Example 14070

Example 14070 Intermediate 1400I (30 mg, 0.015 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (7.48mg, 0.018 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 35-75% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. Thematerial was further purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 3.9 mg, and itsestimated purity by LCMS analysis was 99%. Analysis condition A:Retention time=1.43 min; ESI-MS(+) m/z 1199.4 (M+2H), most abundant ion;Analysis condition B: Retention time=2.57 min; ESI-MS(+) m/z 1199.9(M+2H), most abundant ion. ESI-HRMS(+) m/z: Calculated: 1199.1010 (M+2H)Found: 1199.1018 (M+2H).

Preparation of Example 14071

Intermediate 1400E (39 mg, 0.018 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (8.74mg, 0.021 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 35-75% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 14.4 mg, and its estimated purity by LCMS analysiswas 97%. Analysis condition A: Retention time=1.32 min; ESI-MS(+) m/z874.5 (M+3H); Analysis condition B: Retention time=2.71 min; ESI-MS(+)m/z 874.8 (M+3H); ESI-HRMS(+) m/z:

Calculated: 1310.6489 (M+2H) Found: 1310.6461 (M+2H).

Preparation of Example 14072

Intermediate 1400J (35 mg, 0.017 mmol) and(S)-4-azido-2-palmitamidobutanoic acid (7.68 mg, 0.020 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via preparative LC/MSwith the following conditions: Column: XBridge C18, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 methanol:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 methanol:water with 10-mM ammoniumacetate; Gradient: 55-95% B over 30 minutes, then a 5-minute hold at100% B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 9.0 mg, and its estimated purity by LCMS analysis was 97%. Analysiscondition A: Retention time=1.80 min; ESI-MS(+) m/z 826.1 (M+3H), mostabundant ion; Analysis condition B: Retention time=3.13 min; ESI-MS(+)m/z 1238.8 (M+2H) ESI-HRMS(+) m/z: Calculated: 1238.1483 (M+2H) Found:1238.1484 (M+2H).

Preparation of Example 14073

Intermediate 1400K (30 mg, 0.015 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (7.62mg, 0.018 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 60-100% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 12.0 mg, and its estimated purity by LCMS analysiswas 100%. Analysis condition A: Retention time=1.92 min; ESI-MS(+) m/z1181.0 (M+2H); Analysis condition B: Retention time=3.12 min; ESI-MS(+)m/z 1181.1 (M+2H), most abundant ion; ESI-HRMS(+) m/z: Calculated:1180.5936 (M+2H) Found: 1180.5931 (M+2H).

Preparation of Example 14074

Intermediate 1400L (30 mg, 0.015 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (7.56mg, 0.018 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 45-85% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 25.4 mg, and its estimated purity by LCMS analysiswas 100%. Analysis condition A: Retention time=1.68 min; ESI-MS(+) m/z1189.5 (M+2H); Analysis condition B: Retention time=3.20 min; ESI-MS(+)m/z 1189.5 (M+2H); ESI-HRMS(+) m/z:

Calculated: 1189.0649 (M+2H) Found: 1189.0642 (M+2H).

Preparation of Example 14075

Intermediate 1400J (40 mg, 0.019 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (9.46mg, 0.023 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 40-80% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. Thematerial was further purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 0-35% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 5.7 mg, and itsestimated purity by LCMS analysis was 100%. Analysis condition A:Retention time=1.34 min; ESI-MS(+) m/z 936.2 (M+2H); Analysis conditionB: Retention time=2.97 min; ESI-MS(+) m/z 936.2 (M+2H); ESI-HRMS(+) m/z:Calculated: 960.9769 (M+2H) Found: 960.9749 (M+2H).

Preparation of Example 14076

Intermediate 1300V (30 mg, 0.015 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (7.49mg, 0.018 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 40-80% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Thematerial was further purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile PhaseB: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Gradient:25-65% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20mL/min. Fractions containing the desired product were combined and driedvia centrifugal evaporation. The yield of the product was 2.0 mg, andits estimated purity by LCMS analysis was 100%. Analysis condition A:Retention time=1.43 min; ESI-MS(+) m/z 1198.6 (M+2H), most abundant ion;Analysis condition B: Retention time=2.86 min; ESI-MS(+) m/z 1198.7(M+2H), most abundant ion.

Preparation of Example 14077

Intermediate 1300V (10 mg, 5.04 μmol) and(S)-4-azido-2-palmitamidobutanoic acid (2.32 mg, 6.05 μmol) were reactedas in the general triazole formation procedure above to afford crudeproduct. The crude material was purified via preparative LC/MS with thefollowing conditions: Column: XBridge C18, 19×200 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 25-65% B over 30 minutes, then a 5-minute hold at 100% B;Flow: 20 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 3.2mg, and its estimated purity by LCMS analysis was 100%. Analysiscondition A: Retention time=1.85 min; ESI-MS(+) m/z 1184.2 (M+2H);ESI-HRMS(+) m/z: Calculated: 1003.5215 (M+2H) Found: 1003.5189 (M+2H).

Preparation of Example 14078

Intermediate 1300Y (30 mg, 0.014 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (7.09mg, 0.017 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: watersxbridge c-18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 10-50% B over30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 10.2 mg, and its estimatedpurity by LCMS analysis was 96%. Analysis condition A: Retentiontime=1.28 min; ESI-MS(+) m/z 836.3 (M+3H), most abundant ion; Analysiscondition B: Retention time=2.35 min; ESI-MS(+) m/z 836.8 (M+3H), mostabundant ion.

Preparation of Example 14079

Intermediate 1300Y (10 mg, 4.77 μmol) and(S)-4-azido-2-palmitamidobutanoic acid (2.19 mg, 5.73 μmol) were reactedas in the general triazole formation procedure above to afford crudeproduct. The crude material was purified via preparative LC/MS with thefollowing conditions: Column: waters xbridge c-18, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: 30-70% B over 30 minutes, then a 5-minute hold at100% B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 2.5 mg, and its estimated purity by LCMS analysis was 96%. Analysiscondition A: Retention time=1.50 min; ESI-MS(+) m/z 1239.5 (M+2H), mostabundant ion.

Preparation of Example 14080

Intermediate 1300X (8 mg, 4.06 μmol) and(S)-4-azido-2-palmitamidobutanoic acid (1.87 mg, 4.87 μmol) were reactedas in the general triazole formation procedure above to afford crudeproduct. The crude material was purified via preparative LC/MS with thefollowing conditions: Column: XBridge C18, 19×200 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 25-65% B over 30 minutes, then a 5-minute hold at 100% B;Flow: 20 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 3.4mg, and its estimated purity by LCMS analysis was 96%. Analysiscondition A: Retention time=2.12 min; ESI-MS(+) m/z 1176.7 (M+2H), mostabundant ion.

Preparation of Example 14081

Intermediate 1400K (10 mg, 5.13 μmol) and(S)-4-azido-2-palmitamidobutanoic acid (2.36 mg, 6.16 μmol) were reactedas in the general triazole formation procedure above to afford crudeproduct. The crude material was purified via preparative LC/MS with thefollowing conditions: Column: waters xbridge c-18, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM; ammoniumacetate; Gradient: 35-75% B over 30 minutes, then a 5-minute hold at100% B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation. The yield of the productwas 3.5 mg, and its estimated purity by LCMS analysis was 100%. Analysiscondition A: Retention time=2.44 min; ESI-MS(+) m/z 1166.0 (M+2H), mostabundant ion.

P Preparation of Example 14082

Intermediate 1400J (48 mg, 0.023 mmol) and(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate (17.64 mg, 0.028 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via preparative LC/MSwith the following conditions: Column: waters xbridge c-18, 19×200 mm,5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1%trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1%trifluoroacetic acid; Gradient: 60-100% B over 25 minutes, then a10-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Thematerial was further purified via preparative LC/MS with the followingconditions: Column: XBridge Phenyl, 19×200 mm, 5-μm particles; MobilePhase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; MobilePhase B: 95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient:38-78% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20mL/min. Fractions containing the desired product were combined and driedvia centrifugal evaporation. The yield of the product was 2.6 mg, andits estimated purity by LCMS analysis was 100%. Analysis condition C:Retention time=1.62 min; ESI-MS(+) m/z 912.8 (M+3H), most abundant ion.

Preparation of Example 14083

Intermediate 1300X (26 mg, 0.013 mmol) and(S)-16-((3-azido-1-carboxypropyl)amino)-16-oxohexadecanoic acid (6.54mg, 0.016 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 40-80% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. Thematerial was further purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 30-70% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 3.1 mg, and itsestimated purity by LCMS analysis was 100%. Analysis condition A:Retention time=1.42 min; ESI-MS(+) m/z 1191.8 (M+2H), most abundant ion.Analysis condition B: Retention time=2.86 min; ESI-MS(+) m/z 1191.6(M+2H), most abundant ion.

Preparation of Example 14084

Intermediate 1400L (10 mg, 5.09 μmol) and(S)-4-azido-2-palmitamidobutanoic acid (2.34 mg, 6.11 μmol) were reactedas in the general triazole formation procedure above to afford crudeproduct. The crude material was purified via preparative LC/MS with thefollowing conditions: Column: XBridge C18, 19×200 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 30-70% B over 30 minutes, then a 5-minute hold at 100% B;Flow: 20 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The yield of the product was 2.7mg, and its estimated purity by LCMS analysis was 100%. Analysiscondition A: Retention time=1.87 min; ESI-MS(+) m/z 1175.3 (M+2H), mostabundant ion; Analysis condition B: Retention time=3.10 min; ESI-MS(+)m/z 1175.7 (M+2H), most abundant ion.

Preparation of Example 14085

Intermediate 1300Y (54.8 mg, 0.026 mmol) and(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate (20.12 mg, 0.031 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via preparative LC/MSwith the following conditions: Column: XBridge Phenyl, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 0.1%trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1%trifluoroacetic acid; Gradient: 40-80% B over 30 minutes, then a10-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 15.2 mg, and its estimated purity by LCMSanalysis was 100%. Analysis condition C: Retention time=1.87 min;ESI-MS(+) m/z 912.8 (M+3H), most abundant ion.

Preparation of Example 14086

Intermediate 1400E (48.5 mg, 0.022 mmol) and(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate (16.89 mg, 0.026 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via preparative LC/MSwith the following conditions: Column: XBridge Phenyl, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 0.1%trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1%trifluoroacetic acid; Gradient: 35-75% B over 30 minutes, then a10-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 18.3 mg, and its estimated purity by LCMSanalysis was 100%. Analysis condition C: Retention time=2.90 min;ESI-MS(+) m/z 951.3 (M+3H), most abundant ion.

Preparation of Example 14087

Intermediate 1400K (54 mg, 0.028 mmol) and(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate (21.32 mg, 0.033 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via preparative LC/MSwith the following conditions: Column: XBridge Phenyl, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 0.1%trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1%trifluoroacetic acid; Gradient: 60-100% B over 30 minutes, then a10-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 35.9 mg, and its estimated purity by LCMSanalysis was 100%. Analysis condition C: Retention time=3.23 min;ESI-MS(+) m/z 1295.2 (M+2H), most abundant ion.

Preparation of Example 14088

Intermediate 1400L (54 mg, 0.027 mmol) and(R)-2,5,7,8-tetramethyl-2-((4R,8R)-4,8,12-trimethyltridecyl)chroman-6-yl4-((5-azidopentyl)amino)-4-oxobutanoate (21.13 mg, 0.033 mmol) werereacted as in the general triazole formation procedure above to affordcrude product. The crude material was purified via preparative LC/MSwith the following conditions: Column: waters CSH c-18, 19×200 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 0.1%trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1%trifluoroacetic acid; Gradient: 80-100% B over 30 minutes, then a15-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 29.2 mg, and its estimated purity by LCMSanalysis was 100%. Analysis condition C: Retention time=2.91 min;ESI-MS(+) m/z 869.9 (M+3H), most abundant ion.

Preparation of Example 14089

Intermediate 1300V (13 mg, 6.55 μmol) and3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine (2.060mg, 6.55 μmol), were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:waterwith 0.1% trifluoroacetic acid; Gradient: 15-55% B over 30 minutes, thena 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Thematerial was further purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 1.0 mg, and itsestimated purity by LCMS analysis was 100%. Analysis condition A:Retention time=1.60 min; ESI-MS(+) m/z 1149.3 (M+2H), most abundant ion;Analysis condition B: Retention time=3.13 min; ESI-MS(+) m/z 1149.4(M+2H), most abundant ion; ESI-HRMS(+) m/z: Calculated: 1149.0648 (M+2H)Found: 1149.0635 (M+2H).

Preparation of Example 14090

Intermediate 1300W (12.7 mg, 6.41 μmol) and3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine (2.014mg, 6.41 μmol), were reacted as in the general triazole formationprocedure above to afford crude product. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol:water with10-mM ammonium acetate; Mobile Phase B: 95:5 methanol:water with 10-mMammonium acetate; Gradient: 45-85% B over 30 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. Thematerial was further purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 10-50% Bover 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 1.7 mg, and itsestimated purity by LCMS analysis was 97%. Analysis condition A:Retention time=1.63 min; ESI-MS(+) m/z 1148.9 (M+2H), most abundant ion;Analysis condition B: Retention time=3.20 min; ESI-MS(+) m/z 1148.9(M+2H), most abundant ion.

Preparation of Example 14092

Intermediate 1400J (20 mg, 9.55 μmol) and(S)-1-azido-40-carboxy-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicacid (11.42 mg, 0.011 mmol) were reacted as in the general triazoleformation procedure above to afford crude product. The crude materialwas purified via preparative LC/MS with the following conditions:Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile:water with 0.1% trifluoroacetic acid; Gradient: 20-60% Bover 30 minutes, then a 10-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 4.9 mg, and itsestimated purity by LCMS analysis was 95%. Analysis condition A:Retention time=1.39 min; ESI-MS(+) m/z 1028.8 (M−3H), most abundant ion;Analysis condition B: Retention time=3.08 min; ESI-MS(+) m/z 1028.8(M−3H), most abundant ion; ESI-HRMS(+) m/z: Calculated: 1544.8138 (M+2H)Found: 1544.8114 (M+2H).

Preparation of Example 14093

Intermediate 1300V (30 mg, 15.0 μmol) and(S)-1-azido-40-carboxy-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicacid (18.08 mg, 0.018 mmol) were reacted as in the general triazoleformation procedure above to afford crude product. The crude materialwas purified via preparative LC/MS with the following conditions:Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile:water with 0.1% trifluoroacetic acid; Gradient: 35-75% Bover 30 minutes, then a 7-minute hold at 100% B; Flow: 20 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The material was further purified viapreparative LC/MS with the following conditions: Column: XBridge C18,19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with10-mM ammonium acetate; Gradient: 30-70% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Theyield of the product was 2.6 mg, and its estimated purity by LCMSanalysis was 100%. Analysis condition A: Retention time=1.96 min;ESI-MS(+) m/z 994.3 (M+3H), most abundant ion; ESI-HRMS(+) m/z:

Calculated: 1489.8080 (M+2H) Found: 1489.8043 (M+2H).

Preparation of Example 14095

Intermediate 1300V (80 mg, 40.0 μmol) and(S)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid (17.77mg, 0.040 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude product was purifiedby Prep-HPLC (Column: XBridge Prep C18 30×100 mm Sum, Solvent A=10 mMAmmonium Acetate in 95:5 H2O/ACN, Solvent B=10 mM Ammonium Acetate in5:95 H₂O/ACN. Flow rate: 40 ml/min, 15-50% B, 60 min, Sens=100%). Theyield of the product was 24 mg, and its estimated purity by LCMSanalysis was 90%. Analysis condition E: Retention time=2.51 min;ESI-MS(+) m/z 1213.12 (M+2H), most abundant ion; ESI-HRMS(+) m/z:Calculated: 1212.1452 (M+2H) Found: 1212.1405 (M+2H).

Preparation of Example 14096

Intermediate 1300W (50 mg, 25.0 μmol) and(S)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid (11.11mg, 0.025 mmol) were reacted as in the general triazole formationprocedure above to afford crude product. The crude product was purifiedby Prep-HPLC (Column: XBridge Prep C18 30×100 mm Sum, Solvent A=10 mMAmmonium Acetate in 95:5 H2O/ACN, Solvent B=10 mM Ammonium Acetate in5:95 H₂O/ACN. Flow rate: 40 ml/min, 15-50% B, 60 min, Sens=100%). Theyield of the product was 14 mg, and its estimated purity by LCMSanalysis was 90%. Analysis condition E: Retention time=2.62 min;ESI-MS(+) m/z 1212.31 (M+2H), most abundant ion; ESI-HRMS(+) m/z:Calculated: 1211.6532 (M+2H) Found: 1211.6525 (M+2H).

Preparation of Example 14097

Intermediate 1300W (500 mg, 25.0 μmol) and(S)-1-azido-40-carboxy-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicacid (25.10 mg, 0.025 mmol) were reacted as in the general triazoleformation procedure above to afford crude product. The crude product waspurified by Prep-HPLC (Column: XBridge Prep C18 30×100 mm Sum, SolventA=10 mM Ammonium Acetate in 95:5 H₂O/ACN, Solvent B=10 mM AmmoniumAcetate in 5:95 H2O/ACN. Flow rate: 40 ml/min, 15-50% B, 60 min,Sens=100%). The yield of the product was 11 mg, and its estimated purityby LCMS analysis was 90%. Analysis condition E: Retention time=2.57 min;ESI-MS(+) m/z 1489.99 (M+2H), most abundant ion; ESI-HRMS(+) m/z:Calculated: 1489.3160 (M+2H).

Found: 1489.3155 (M+2H).

Preparation of Example 14098

Intermediate 1300V (50 mg, 25.0 μmol) and(S)-1-azido-16-carboxy-13,18-dioxo-3,6,9-trioxa-12,17-diazapentatriacontan-35-oicacid (16.23 mg, 0.025 mmol) were reacted as in the general triazoleformation procedure above to afford crude product. The crude product waspurified by Prep-HPLC (Column: XBridge Prep C18 30×100 mm Sum, SolventA=10 mM Ammonium Acetate in 95:5 H2O/ACN, Solvent B=10 mM AmmoniumAcetate in 5:95 H2O/ACN. Flow rate: 40 ml/min, 15-50% B, 60 min,Sens=100%). The yield of the product was 29 mg, and its estimated purityby LCMS analysis was 90%. Analysis condition E: Retention time=2.50 min;ESI-MS(+) m/z 1314.37 (M+2H), most abundant ion ESI-HRMS(+) m/z:Calculated: 1313.7031 (M+2H) Found: 1313.7031 (M+2H).

Preparation of Example 14099

Intermediate 1300V (50 mg, 25.0 μmol) and(S)-1-azido-28-carboxy-25,30-dioxo-3,6,9,12,15,18,21-heptaoxa-24,29-diazaheptatetracontan-47-oicacid (20.67 mg, 0.025 mmol) were reacted as in the general triazoleformation procedure above to afford crude product. The crude product waspurified by Prep-HPLC (Column: XBridge Prep C18 30×100 mm Sum, SolventA=10 mM Ammonium Acetate in 95:5 H2O/ACN, Solvent B=10 mM AmmoniumAcetate in 5:95 H2O/ACN. Flow rate: 40 ml/min, 15-50% B, 60 min,Sens=100%). The yield of the product was 51 mg, and its estimated purityby LCMS analysis was 90%. Analysis condition E: Retention time=2.52 min;ESI-MS(+) m/z 1402.83 (M+2H), most abundant ion ESI-HRMS(+) m/z:Calculated: 1401.7555 (M+2H) Found: 1401.7561 (M+2H).

Preparation of Example 14100

Intermediate 1300V (50 mg, 25.0 μmol) and(S)-1-azido-22-carboxy-19,24-dioxo-3,6,9,12,15-pentaoxa-18,23-diazahentetracontan-41-oicacid (18.45 mg, 0.025 mmol) were reacted as in the general triazoleformation procedure above to afford crude product.

The crude product was purified by Prep-HPLC (Column: XBridge Prep C1830×100 mm Sum, Solvent A=10 mM Ammonium Acetate in 95:5 H₂O/ACN, SolventB=10 mM Ammonium Acetate in 5:95 H₂O/ACN. Flow rate: 40 ml/min, 15-50%B, 60 min, Sens=100%). The yield of the product was 26 mg, and itsestimated purity by LCMS analysis was 90%. Analysis condition D:Retention time=2.25 min; ESI-MS(+) m/z 1358.8 (M+2H), most abundant ionESI-HRMS(+) m/z:

Calculated: 1357.7293 (M+2H) Found: 1357.7263 (M+2H).

Preparation of Example 14101

Intermediate 1300V (57 mg, 29.0 μmol) and(S)-1-azido-15-carboxy-13,17-dioxo-3,6,9-trioxa-12,16-diazatetratriacontane-34-oicacid (18.10 mg, 0.029 mmol) were reacted as in the general triazoleformation procedure above to afford crude product. The crude product waspurified by Prep-HPLC (Column: XBridge Prep C18 30×100 mm Sum, SolventA=10 mM Ammonium Acetate in 95:5 H₂O/ACN, Solvent B=10 mM AmmoniumAcetate in 5:95 H₂O/ACN. Flow rate: 40 ml/min, 15-50% B, 60 min,Sens=100%). The yield of the product was 15 mg, and its estimated purityby LCMS analysis was 90%. Analysis condition E: Retention time=2.50 min;ESI-MS(+) m/z 1307.4 (M+2H), most abundant ion; ESI-HRMS(+) m/z:Calculated: 1306.6953 (M+2H) Found: 1306.6927 (M+2H).

Preparation of Example 14102

Intermediate 1300V (75 mg, 38.0 μmol) and(S)-1-azido-39-carboxy-37,41-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,40-diazaoctapentacontan-58-oicacid (37.1 mg, 0.038 mmol) were reacted as in the general triazoleformation procedure above to afford crude product. The crude product waspurified by Prep-HPLC (Column: XBridge Prep C18 30×100 mm Sum, SolventA=10 mM Ammonium Acetate in 95:5 H2O/ACN, Solvent B=10 mM AmmoniumAcetate in 5:95 H₂O/ACN. Flow rate: 40 ml/min, 15-50% B, 60 min,Sens=100%). The yield of the product was 25 mg, and its estimated purityby LCMS analysis was 90%. Analysis condition D: Retention time=2.31 min;ESI-MS(+) m/z 1483.4 (M+2H), most abundant ion. ESI-HRMS(+) m/z:Calculated: 988.8692 (M+3H).

Found: 988.8696 (M+3H).

Preparation of Example 14103

Intermediate 1300V (75 mg, 38.0 μmol) and(S)-1-azido-39-carboxy-37,41-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,40-diazaoctapentacontan-58-oicacid (37.1 mg, 0.038 mmol), were reacted as in the general triazoleformation procedure above to afford crude product. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Xbridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 10 mmAmmonium Acetate in 95:5 H₂O:ACN; Mobile Phase B: 10 mm Ammonium Acetatein 5:95 H2O:ACN; Gradient: 15-50% B over 60 minutes, Flow: 40 mL/min,Sens: 100%. Fractions containing the desired product were combined anddried via centrifugal evaporation. The yield of the product was 25 mg,and its estimated purity by LCMS analysis was 90%. Analysis condition D:Retention time=2.243 min; ESI-MS(+) m/z 1483.4 (M+2H), most abundantion; ESI-HRMS(+) m/z: Calculated: 1482.8001 (M+2H).

Found: 1482.7974 (M+2H).

Preparation of Example 14104

Intermediate 130AI (75 mg, 37.0 μmol) and(S)-1-azido-40-carboxy-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicacid (37.2 mg, 0.037 mmol), were reacted as in the general triazoleformation procedure above to afford crude product. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Xbridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 10 mmAmmonium Acetate in 95:5 H₂O:ACN; Mobile Phase B: 10 mm Ammonium Acetatein 5:95 H₂O:ACN; Gradient: 15-50% B over 60 minutes, Flow: 40 mL/min,Sens: 100%. Fractions containing the desired product were combined anddried via centrifugal evaporation. The yield of the product was 33 mg,and its estimated purity by LCMS analysis was 90%. Analysis condition D:Retention time=2.27 min; ESI-MS(+) m/z 1002.5 (M+3H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 1502.8163 (M+2H).

Found: 1502.8137.

Preparation of Example 14105

Intermediate 130AK (75 mg, 37.0 μmol) and(S)-1-azido-40-carboxy-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicacid (36.9 mg, 0.037 mmol), were reacted as in the general triazoleformation procedure above to afford crude product. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Xbridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 10 mmAmmonium Acetate in 95:5 H₂O:ACN; Mobile Phase B: 10 mm Ammonium Acetatein 5:95 H₂O:ACN; Gradient: 15-50% B over 60 minutes, Flow: 40 mL/min,Sens: 100%. Fractions containing the desired product were combined anddried via centrifugal evaporation. The yield of the product was 31 mg,and its estimated purity by LCMS analysis was 90%. Analysis condition D:Retention time=2.27 min; ESI-MS(+) m/z 1007.2 (M+3H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 1509.8242 (M+2H).

Found: 1509.8224.

Preparation of Example 14106

Intermediate 130AJ (75 mg, 37.0 μmol) and(S)-1-azido-40-carboxy-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicacid (36.9 mg, 0.037 mmol), were reacted as in the general triazoleformation procedure above to afford crude product. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Xbridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 10 mmAmmonium Acetate in 95:5 H₂O:ACN; Mobile Phase B: 10 mm Ammonium Acetatein 5:95 H₂O:ACN; Gradient: 15-50% B over 60 minutes, Flow: 40 mL/min,Sens: 100%. Fractions containing the desired product were combined anddried via centrifugal evaporation. The yield of the product was 26 mg,and its estimated purity by LCMS analysis was 90%. Analysis condition D:Retention time=2.24 min; ESI-MS(+) m/z 1007.8 (M+3H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 1510.7956 (M+2H).

Found: 1510.7940.

Preparation of Example 14107

Intermediate 130AL (75 mg, 37.0 μmol) and(S)-1-azido-40-carboxy-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicacid (36.6 mg, 0.037 mmol), were reacted as in the general triazoleformation procedure above to afford crude product. The crude materialwas purified via preparative LC/MS with the following conditions:Column: Xbridge C18, 30×100 mm, 5-μm particles; Mobile Phase A: 10 mmAmmonium Acetate in 95:5 H₂O:ACN; Mobile Phase B: 10 mm Ammonium Acetatein 5:95 H2O:ACN; Gradient: 15-50% B over 60 minutes, Flow: 40 mL/min,Sens: 100%. Fractions containing the desired product were combined anddried via centrifugal evaporation. The yield of the product was 29 mg,and its estimated purity by LCMS analysis was 90%. Analysis condition D:Retention time=2.23 min; ESI-MS(+) m/z 1012.5 (M+3H), most abundant ion;ESI-HRMS(+) m/z: Calculated: 1517.8034 (M+2H).

Found: 1517.8015.

Preparation of INT-1400M

The above peptide was synthesized on a 1.0 mmol scale according to theprocedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr(Me)-Pip-Asp-Pro-Dap-Gln-Hyp-Trp-Dab-Trp′-mNle-mNle-Glu-Cys-Gly-[(S)-propargylglycine]

After deprotection and cyclization according to the procedures above,the compound was purified as follows:

The crude material was purified via preparative LC/MS with the followingconditions: Column: XBridge C18, 30×200 mm, 5-μm particles; Mobile PhaseA: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B:95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 15-55% Bover 30 minutes, then a 7-minute hold at 100% B; Flow: 50 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation. The yield of the product was 30 mg, and itsestimated purity by LCMS analysis was 66.9%.

Analysis condition E: Retention time=2.55 min; ESI-MS(−) m/z 1026.7(M−2H), most abundant ion.

Preparation of INT-1400N

The above peptide was synthesized on a 1.8 mmol scale according to theprocedures above. The underlined steps employed the double-couplingprocedure.ClAc-Tyr(4-F)-Pip-Asp-Pro-Dap-Leu-Hyp-Trp-Dab-Trp′-mNle-mNle-Leu-Cys-Gly-[(S)-propargylglycine]

After deprotection and cyclization according to the procedures above,the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 30×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 15-55% B over 30 minutes, then a5-minute hold at 100% B; Flow: 50 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation.

The yield of the product was 90 mg, and its estimated purity by LCMSanalysis was 52.5%. Analysis condition E: Retention time=1.73 min;ESI-MS(+) m/z 1007.0 (M+2H), most abundant ion.

Preparation of Example 14121

Intermediate 1400M (30 mg, 7.59 μmol) and(S)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid (3.34mg, 7.59 μmol) were reacted as in the general triazole formationprocedure to afford crude product. The crude material was purified viapreparative LC/MS with the following conditions: (Column: XBridge PrepC18 30×100 mm Sum, Solvent A=10 mM Ammonium Acetate in 95:5 H₂O/ACN,Solvent B=10 mM Ammonium Acetate in 5:95 H₂O/ACN. Flow rate: 40 ml/min,15-50% B, 60 min). Fractions containing the desired product werecombined and dried via centrifugal evaporation.

The yield of the product was 8.0 mg, and its estimated purity by LCMSanalysis was 97%.Analysis condition C: Retention time=1.15 min; ESI-MS(+) m/z 832.9(M+3H)Analysis condition D: Retention time=2.29 min; ESI-MS(+) m/z 1248.4(M+2H)Analysis condition E: Retention time=2.45 min; ESI-MS(−) m/z 830.0(M−3H)

ESI-HRMS(+) m/z: Calculated: 1248.1194 (M+2H) Found: 1248.1185 (M+2H).Preparation of Example 14122

Intermediate 1400N (90 mg, 23 μmol) and(S)-18-((3-azido-1-carboxypropyl)amino)-18-oxooctadecanoic acid (10.34mg, 23 μmol) were reacted as in the general triazole formation procedureto afford crude product. The crude material was purified via preparativeLC/MS with the following conditions: (Column: XBridge Prep C18 30×100 mm5 um, Solvent A=10 mM Ammonium Acetate in 95:5 H2O/ACN, Solvent B=10 mMAmmonium Acetate in 5:95 H₂O/ACN. Flow rate: 40 ml/min, 15-50% B, 60min). Fractions containing the desired product were combined and driedvia centrifugal evaporation.

The yield of the product was 12.0 mg, and its estimated purity by LCMSanalysis was 97%.Analysis condition D: Retention time=2.47 min; ESI-MS(+) m/z 1227.2(M+2H)Analysis condition E: Retention time=2.45 min; ESI-MS(+) m/z 1227.2(M+2H)

ESI-HRMS(+) m/z: Calculated: 1226.6429 (M+2H) Found: 1226.6408 (M+2H).Preparation of 1-tert-butyl 18-(perfluorophenyl) octadecanedioate

To a solution of 18-(tert-butoxy)-18-oxooctadecanoic acid (5.00 g, 13.49mmol) in DMF (54.0 ml) was added pyridine (3.82 ml, 47.2 mmol), followedby pentafluorophenyl trifluoroacetate (5.81 ml, 33.7 mmol). A gelformed, and an additional stir bar was added to the reaction mixture.The mixture was stirred vigorously overnight. The reaction mixture wasfiltered (Buchner funnel/paper) to afford a white solid, which washedwith a small amount of DMF. A nitrogen-rich atmosphere was suckedthrough the filter cake for a few hours to provide 1-tert-butyl18-(perfluorophenyl) octadecanedioate (6.50 g, 11.63 mmol, 90% yield).Analysis condition D: Retention time=4.12 min; ESI-MS(+) m/z 559.1(M+Na).

Preparation of Preparation of(S)-5-(benzyloxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid

The mixture of (S)-4-amino-5-(benzyloxy)-5-oxopentanoic acid (1.1 g,4.64 mmol), 1-tert-butyl 18-(perfluorophenyl) octadecanedioate (2.488 g,4.64 mmol) and Hunig's Base (1.053 ml, 6.03 mmol) in DMF (15.45 ml) wasstirred for 24 hr at rt. After 24 hr the reaction was homogeneous. Thereaction mixture was poured into a saturated citric acid solution, andextracted with CH₂Cl₂ (3×50 ml). The organic fractions were combined,washed with brine, dried over Na₂SO₄ and evaporated in vacuo. Theresulting crude product was purified by Biotage (SG, 300 g, 0 to 25%acetone/CH₂Cl₂) to get(S)-5-(benzyloxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (1.31 g, 2.221 mmol, 47.9% yield). Analysis condition D: Retentiontime=2.99 min; ESI-MS(+) m/z 591.2 (M+1); ¹H NMR (500 MHz, METHANOL-d₄)δ 7.41-7.28 (m, 5H), 5.22-5.14 (m, 2H), 4.49 (dd, 5.3 Hz, 1H), 2.42-2.35(m, 2H), 2.27-2.19 (m, 4H), 2.19-2.13 (m, 1H), 1.96 (ddt, J=14.2, 9.0,7.1 Hz, 1H), 1.65-1.52 (m, 4H), 1.48-1.41 (m, 9H), 1.37 (br. s., 1H),1.33-1.26 (m, 24H).

Preparation of (S)-1-benzyl 5-(perfluorophenyl)2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanedioate

To a 100 ml RBF was added(S)-5-(benzyloxy)-4-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (1.31 g, 2.221 mmol), N,N-Dimethylformamide (7.40 ml), pyridine(0.387 g, 4.89 mmol), and perfluorophenyl 2,2,2-trifluoroacetate (1.244g, 4.44 mmol). The reaction was allowed to stir for 24 hr at it After 24hr the reaction mixture was poured into a saturated citric acidsolution, and extracted with CH₂Cl₂ (3×50 ml). The organic fractionswere combined, washed with brine, dried over Na₂SO₄ and evaporated invacuo. The crude oil (S)-1-benzyl 5-(perfluorophenyl)2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanedioate was used as is inthe next step.

Analysis condition D: Retention time=3.60 min; ESI-MS(+) m/z 757.2(M+1).

Preparation of (S)-tert-butyl11-((benzyloxy)carbonyl)-1-(9H-fluoren-9-yl)-3,8,13-trioxo-2-oxa-4,7,12-triazatriacontan-30-oate

The mixture of (9H-fluoren-9-yl)methyl (2-aminoethyl)carbamate, HCl(0.850 g, 2.67 mmol), (S)-1-benzyl 5-(perfluorophenyl)2-(18-(tert-butoxy)-18-oxooctadecanamido)pentanedioate (1.679 g, 2.221mmol) and Hunig's Base (1.164 ml, 6.66 mmol) in DMF (10 ml) was stirredfor 24 hr at rt. After 24 hr the reaction was homogeneous. The reactionmixture was poured into a saturated citric acid solution, and extractedwith CH₂Cl₂ (3×50 ml). The organic fractions were combined, washed withbrine, dried over Na₂SO₄ and evaporated in vacuo. The crude product wastriturated with MeCN to get (S)-tert-butyl11-((benzyloxy)carbonyl)-1-(9H-fluoren-9-yl)-3,8,13-trioxo-2-oxa-4,7,12-triazatriacontan-30-oate(1.448 g, 1.695 mmol, 76.3% yield).

Analysis condition D: Retention time=3.61 min; ESI-MS(+) m/z 855.5 (M+1)¹H NMR (500 MHz, METHANOL-d₄) δ 7.81 (d, J=7.6 Hz, 2H), 7.66 (d, J=7.5Hz, 2H), 7.40 (t, J=7.5 Hz, 2H), 7.37-7.24 (m, 7H), 5.12 (d, J=2.3 Hz,2H), 4.49-4.42 (m, 1H), 4.33 (dd, J=6.9, 4.4 Hz, 2H), 4.24-4.16 (m, 1H),3.26-3.14 (m, 4H), 2.34-2.13 (m, 7H), 1.93 (dt, J=9.5, 6.9 Hz, 1H),1.66-1.52 (m, 4H), 1.46 (s, 9H), 1.37-1.20 (m, 24H).

Preparation of(S)-5-((2-aminoethyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid

The mixture of (S)-tert-butyl11-((benzyloxy)carbonyl)-1-(9H-fluoren-9-yl)-3,8,13-trioxo-2-oxa-4,7,12-triazatriacontan-30-oate(1.448 g, 1.695 mmol) in Methanol (28.3 ml) was added PALLADIUM ONCARBON (0.180 g, 0.170 mmol). The flask was sealed with a septum andcharged with HYDROGEN via a balloon. The next day the reaction wasfiltered through celite to remove the catalyst and the the filtrate wasevaporated in vacuo to afford(S)-5-((2-aminoethyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid which is used as is. Analysis condition D: Retention time=2.44 min;ESI-MS(+) m/z 542.2 (M+1)

Preparation of(S)-5-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid

(S)-5-((2-aminoethyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (0.918 g, 1.694 mmol) in DCM (42.4 ml) at 0° C. was added DIEA(0.888 ml, 5.08 mmol) and 9-FLUORENYLMETHYL CHLOROFORMATE (0.482 g,1.864 mmol). The resulting solution was allowed to warmed to rt stirredovernight. The crude product was purified by Prep-HPLC (Solvent A=10%Acetonitrile—90% H2O—0.1% TFA, Solvent B=90% Acetonitrile—10% H2O—0.1%TFA. Column: waters-sunFire OBD30×100 mm, S10, Flow rate: 40 ml/min,45-100% B, 10 min, additional 6 min after 100% B) to get(S)-5-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (443 mg, 0.580 mmol, 34.2% yield), 2 steps.

Analysis condition C: Retention time=1.84 min; ESI-MS(+) m/z 764.5 (M+1)¹H NMR (500 MHz, METHANOL-d₄) δ 7.81 (d, J=7.5 Hz, 2H), 7.67 (d, J=7.5Hz, 2H), 7.40 (t, J=7.5 Hz, 2H), 7.37-7.29 (m, 2H), 4.42 (dd, J=9.2, 4.8Hz, 1H), 4.35 (dd, J=6.9, 4.0 Hz, 2H), 4.28-4.17 (m, 1H), 3.30-3.18 (m,4H), 2.36-2.12 (m, 7H), 2.02-1.91 (m, 1H), 1.69-1.52 (m, 4H), 1.46 (s,9H), 1.40-1.20 (m, 24H).

Preparation of modified chlorotrityl resin 14A

To a mixture of(S)-5-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)amino)-2-(18-(tert-butoxy)-18-oxooctadecanamido)-5-oxopentanoicacid (443 mg, 0.580 mmol) and Hunig's Base (658 μl, 3.77 mmol) dissolvedin CH₂Cl₂ (5857 μl) was added 4-CHLOROTOLUENE (73.4 mg, 0.580 mmol).LC/MS of this mixture was taken as the standard. Swelled1-chloro-2-(chloro(phenyl)(p-tolyl)methyl)benzene (1160 mg, 1.855 mmol)resin with CH₂Cl₂ (1.17E+04 μl) then add solution of acid and DIEA.Shaked the resin (monitor by taking aliquot out and taking LC/MS tocompare to the standard LC/MS run) for 45 min. Added 25 mL of 9:1MeOH/DIEA to the reaction vessel and immediately filtered the resin. Theresin was rinsed 3 times with DCM (stirring for ˜20 s in betweenwashes). The resin was then shaken with ˜20 mL DMF for 5 min, thenfiltered. This was repeated 2 more times with DMF, then 3 times withDCM. The resin was dried on the fitted funnel with N₂ being passedthrough the resin.Final weight: 1.5065 g, Theo. loading: 260 mg/0.1 mmol, Calc. loading:371 mg/0.1 mmol (based on 70% yield).

Preparation of Example 14123

Example 14123 was synthesized on a 0.2 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr(4-F)-Pip-Asp-Pro-Dap-Leu-Hyp-Trp-Dab-Trp′-mNle-mNle-Leu-Cys-Gly-((S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoicacid)-[Modified resin 14A]. After deprotection following GlobalDeprotection procedure B and cyclization according to Cyclization methodB, the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with10-mM ammonium acetate; Gradient: 25-65% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Thematerial was further purified via preparative LC/MS with the followingconditions: Column: Waters CSH C18, 19×200 mm, 5-μm particles; MobilePhase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; MobilePhase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid;Gradient: 10-100% B over 15 minutes, then a 5-minute hold at 100% B;Flow: 20 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation.

The yield of the product was 1.4 mg, and its estimated purity by LCMSanalysis was 96%.Analysis condition C: Retention time=2.02 min; ESI-MS(+) m/z 838.7 (M+3)ESI-HRMS(+) m/z: Calculated: 1257.1564 (M+2H) Found: 1257.1641 (M+2H).

Preparation of Example 14124

Example 14123 was synthesized on a 0.2 mmol scale according to thegeneral procedures above, including chloroacetic acid coupling procedureA. The underlined steps employed the double-coupling procedure, anditalicized residues were coupled with a 30 min single coupling.ClAc-Tyr(4-F)-Pip-Asp-Pro-Dap-Leu-Hyp-Trp-Dab-Trp′-mNle-mNle-Leu-Cys-Gly-((S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoicacid)-[Modified resin 14A]. After deprotection following GlobalDeprotection procedure B and cyclization according to Cyclization methodB, the compound was purified as follows: The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM ammonium acetate; Gradient: 25-65% B over 30 minutes, then a5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation. Thematerial was further purified via preparative LC/MS with the followingconditions: Column: Waters CSH C18, 19×200 mm, 5-μm particles; MobilePhase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; MobilePhase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid;Gradient: 10-100% B over 15 minutes, then a 5-minute hold at 100% B;Flow: 20 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The material was further purifiedvia preparative LC/MS with the following conditions: Column: Waters CSHC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:waterwith 0.1% trifluoroacetic acid; Gradient: 20-60% B over 30 minutes, thena 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation.

The yield of the product was 7.8 mg, and its estimated purity by LCMSanalysis was 93%.Analysis condition E: Retention time=1.90 min; ESI-MS(+) m/z 1250.9(M+2) ESI-HRMS(+) m/z: Calculated: 1250.1486 (M+2H) Found: 1250.1547(M+2H).

Preparation of 4-(18-(tert-butoxy)-18-oxooctadecanamido)butanoic acid

The mixture of 4-aminobutanoic acid (250 mg, 2.424 mmol), 1-tert-butyl18-(perfluorophenyl) octadecanedioate (1301 mg, 2.424 mmol) and Hunig'sBase (550 μl 3.15 mmol) in DMF (8081 μl) was stirred for 24 hr at rt.After 24 hr the reaction was homogeneous. The reaction mixture waspoured into a saturated citric acid solution, and extracted with CH₂Cl₂(3×50 ml). The organic fractions were combined, washed with brine, driedover Na₂SO₄ and evaporated in vacuo. The crude product4-(18-(tert-butoxy)-18-oxooctadecanamido)butanoic acid was used as is.

Analysis condition D: Retention time=2.02 min; ESI-MS(+) m/z 456.2 (M+1)

Preparation of tert-butyl1-azido-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oate

To a solution of 4-(18-(tert-butoxy)-18-oxooctadecanamido)butanoic acid(0.552 g, 1.212 mmol) in DMF (12.12 ml) was added Hunig's Base (0.635ml, 3.64 mmol) and HATU (0.922 g, 2.424 mmol).35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontan-1-amine(0.692 g, 1.212 mmol) was then added, and the solution stirred at rt.The mixture was stirred overnight. The reaction mixture was poured intoa saturated citric acid solution, and extracted with CH₂Cl₂ (3×50 ml).The organic fractions were combined, washed with brine, dried overNa₂SO₄ and evaporated in vacuo. The crude product tert-butyl1-azido-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oatewas used as is.

Analysis condition D: Retention time=2.96 min; ESI-MS(+) m/z 1008.8(M+1).

Preparation of1-azido-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicacid

The mixture of tert-butyl1-azido-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oate(1.222 g, 1.212 mmol) and TFA (3.0 ml, 38.9 mmol) in DCM (10 ml) wasstirred at rt overnight. The reaction mixture was poured into a bine,and extracted with CH₂Cl₂ (3×50 ml). The organic fractions were combinedand evaporated in vacuo. The resulting crude product was purified byPrep-HPLC (Solvent A=10% MeOH—90% H2O—0.1% TFA, Solvent B=90% MeOH—10%H2O—0.1% TFA. Column: PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40ml/min, 55-100% B, 10 min, stop at 13 min) to obtain1-azido-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicacid (696 mg, 0.731 mmol, 60.3% yield), 3 steps. Analysis condition C:Retention time=1.24 min; ESI-MS(+) m/z 952.6 (M+1) ¹H NMR (500 MHz,CHLOROFORM-d) δ 6.90 (t, J=5.0 Hz, 1H), 6.69-6.58 (m, 1H), 3.74-3.52 (m,44H), 3.44 (q, J=5.2 Hz, 2H), 3.39 (t, J=5.1 Hz, 2H), 3.29 (q, J=6.0 Hz,2H), 2.30 (dt, J=17.9, 7.2 Hz, 4H), 2.23-2.09 (m, 2H), 1.84 (quin, J=6.6Hz, 2H), 1.61 (sxt, J=7.7 Hz, 4H), 1.39-1.17 (m, 24H).

Preparation of Example 14125

Intermediate 1300V (151 mg, 76 μmol) and1-azido-37,42-dioxo-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36,41-diazanonapentacontan-59-oicacid (72.5 mg, 76 μmol) were reacted as in the general triazoleformation procedure to afford crude product. The crude material waspurified via preparative LC/MS with the following conditions: Column:XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile:water with 10-mM ammonium acetate; Gradient: 32-54% B over14 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation.

The yield of the product was 45.8 mg, and its estimated purity by LCMSanalysis was 95%.Analysis condition D: Retention time=2.47 min; ESI-MS(+) m/z 979.5(M+3H)Analysis condition E: Retention time=2.01 min; ESI-MS(+) m/z 979.4(M+3H)

ESI-HRMS(+) m/z: Calculated: 1467.8131 (M+2H) Found: 1467.8079 (M+2H).Preparation of tert-butyl 18-((3-azidopropyl)amino)-18-oxooctadecanoate

The mixture of 3-azidopropan-1-amine (250 mg, 2.497 mmol), 1-tert-butyl18-(perfluorophenyl) octadecanedioate (1340 mg, 2.497 mmol) and Hunig'sBase (567 μl, 3.25 mmol) in DMF (8323 μl) was stirred for 24 hr at rt.After 24 hr the reaction was homogeneous. The reaction mixture waspoured into a saturated citric acid solution, and extracted with CH₂Cl₂(3×50 ml). The organic fractions were combined, washed with brine, driedover Na₂SO₄ and evaporated in vacuo. The crude product tert-butyl18-((3-azidopropyl)amino)-18-oxooctadecanoate was used as is.

Analysis condition D: Retention time=2.76 min; ESI-MS(+) m/z 475.1(M+Na).

Preparation of 18-((3-azidopropyl)amino)-18-oxooctadecanoic acid

The mixture of tert-butyl 18-((3-azidopropyl)amino)-18-oxooctadecanoate(750 mg, 1.657 mmol) and TFA (3.0 ml, 38.9 mmol) in DCM (10 ml) wasstirred at rt for 2 h. The resulting crude product was purified byPrep-HPLC (Solvent A=10% MeOH—90% H₂O—0.1% TFA, Solvent B=90% MeOH—10%H₂O—0.1% TFA. Column: PHENOMENEX LUNA 30×100 mm, S10, Flow rate: 40ml/min, 50-100% B, 10 min, stop at 13 min) to obtain18-((3-azidopropyl)amino)-18-oxooctadecanoic acid (138 mg, 0.348 mmol,21.00% yield), 2 steps. Analysis condition D: Retention time=2.67 min;ESI-MS(+) m/z 419.1 (M+Na)¹H NMR (500 MHz, CHLOROFORM-d) δ 7.28 (s, 1H),3.47-3.28 (m, 4H), 2.40-2.27 (m, 2H), 2.24-2.14 (m, 2H), 1.87-1.76 (m,2H), 1.65 (dq, J=14.9, 7.7 Hz, 4H), 1.40-1.18 (m, 24H).

Preparation of Example 14126

Intermediate 1300V (170 mg, 86 μmol) and18-((3-azidopropyl)amino)-18-oxooctadecanoic acid (34 mg, 86 μmol) werereacted as in the general triazole formation procedure to afford crudeproduct. The crude material was purified via preparative LC/MS with thefollowing conditions: Column: XBridge C18, 19×200 mm, 5-μm particles;Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate;Gradient: 28-56% B over 22 minutes, then a 5-minute hold at 100% B;Flow: 20 mL/min. Fractions containing the desired product were combinedand dried via centrifugal evaporation. The material was further purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:waterwith 0.1% trifluoroacetic acid; Gradient: 25-65% B over 30 minutes, thena 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing thedesired product were combined and dried via centrifugal evaporation.

The yield of the product was 20.3 mg, and its estimated purity by LCMSanalysis was 97%.Analysis condition D: Retention time=2.41 min; ESI-MS(+) m/z 1190.7(M+2H)Analysis condition E: Retention time=2.02 min; ESI-MS(+) m/z 1190.4(M+2H)

ESI-HRMS(+) m/z: Calculated: 1190.1503 (M+2H) Found: 1190.1474 (M+2H).Methods for Testing the Ability of Macrocyclic Peptides to Compete forthe Binding of PD-1 to PD-L1 Using Homogenous Time-Resolved Fluorescence(HTRF) Binding Assays

The ability of the macrocyclic peptides of the present disclosure tobind to PD-L1 was investigated using a PD-1/PD-L1 HomogenousTime-Resolved Fluorescence (HTRF) binding assay.

Methods

Homogenous Time-Resolved Fluorescence (HTRF) Assays of Binding ofSoluble PD-1 to Soluble PD-L1. Soluble PD-1 and soluble PD-L1 refers toproteins with carboxyl-end truncations that remove thetransmembrane-spanning regions and are fused to heterologous sequences,specifically the Fc portion of the human immunoglobulin G sequence (Ig)or the hexahistidine epitope tag (His). All binding studies wereperformed in an HTRF assay buffer consisting of dPBS supplemented with0.1% (w/v) bovine serum albumin and 0.05% (v/v) Tween-20. For thePD-1-Ig/PD-L1-His binding assay, inhibitors were pre-incubated withPD-L1-His (10 nM final) for 15m in 4 μl of assay buffer, followed byaddition of PD-1-Ig (20 nM final) in 1 μl of assay buffer and furtherincubation for 15m. PD-L1 fusion proteins from either human, cynomolgousmacaques, mouse, or other species were used. HTRF detection was achievedusing europium crypate-labeled anti-Ig monoclonal antibody (1 nM final)and allophycocyanin (APC) labeled anti-His monoclonal antibody (20 nMfinal). Antibodies were diluted in HTRF detection buffer and 5 μl wasdispensed on top of binding reaction. The reaction was allowed toequilibrate for 30 minutes and signal (665 nm/620 nm ratio) was obtainedusing an EnVision fluorometer. Additional binding assays wereestablished between PD-1-Ig/PD-L2-His (20 and 5 nM, respectively),CD8O-His/PD-L1-Ig (100 and 10 nM, respectively) and CD80-His/CTLA4-Ig(10 and 5 nM, respectively). Binding/competition studies betweenbiotinylated Compound No. 71 and human PD-L1-His were performed asfollows. Macrocyclic peptide inhibitors were pre-incubated withPD-L1-His (10 nM final) for 60 minutes in 4 μl of assay buffer followedby addition of biotinylated Compound No. 71 (0.5 nM final) in 1 μl ofassay buffer. Binding was allowed to equilibrate for 30 minutes followedby addition of europium crylated labeled Streptavidin (2.5 pM final) andAPC-labeled anti-His (20 nM final) in 5 μl of HTRF buffer. The reactionwas allowed to equilibrate for 30m and signal (665 nm/620 nm ratio) wasobtained using an EnVision fluorometer.

Recombinant Proteins. Carboxyl-truncated human PD-1 (amino acids 25-167)with a C-terminal human Ig epitope tag [hPD-1 (25-167)-3S-IG] and humanPD-L1 (amino acids 18-239) with a C-terminal His epitope tag[hPD-L1(19-239)-tobacco vein mottling virus protease cleavage site(TVMV)-His] were expressed in HEK293T cells and purified sequentially byrecombinant Protein A affinity chromatography and size exclusionchromatography. Human PD-L2-His (Sino Biologicals), CD8O-His (SinoBiologicals), CTLA4-Ig (RnD Systems) were all obtained throughcommercial sources.

Sequence of Recombinant Human PD-1-Ig hPD1(25-167)-3S-IG (SEQ ID NO. 1)  1 LDSPDRPWNP PTFSPALLVV TEGDNATFTC     SFSNTSESFV LNWYRMSPSN 51 QTDKLAAFPE DRSQPGQDCR FRVTQLPNGR     DFHMSVVRAR RNDSGTYLCG101 AISLARKAQI KESLRAELRV TERRAEVPTA     HPSPSPRPAG QFQGSPGGGG151 GREPKSSDKT HTSPPSPAPE LLGGSSVFLF     PPKPKDTLMI SRTPEVTCVV201 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE     EQYNSTYRVV SVLTVLHQDW251 LNGKEYKCKV SNKALPAPIE KTISKAYGQP     REPQVYTLPP SRDELTKNQV301 SLTCLVKGFY PSDIAVEWES NGQPENNYKT     TPPVLDSDGS FFLYSKLTVD351 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL     SPGKSequence of Recombinant Human PD-L1-TVMV-His (PD-L1-His)hPDL1(19-239)-TVMV-His (SEQ ID NO: 2)  1 FTVTVPKDLY VVEYGSNMTI ECKFPVEKQL     DLAALIVYWE MEDKNIIQFV 51 HGEEDLKVQH SSYRQRARLL KDQLSLGNAA     LQITDVKLQD AGVYRCMISY101 GGADYKRITV KVNAPYNKIN QRILVVDPVT     SEHELTCQAE GYPKAEVIWT151 SSDHQVLSGK TTTTNSKREE KLFNVTSTLR     INTTTNEIFY CTFRRLDPEE201 NHTAELVIPE LPLAHPPNER TGSSETVRFQ     GHHHHHH

The results are shown in Table 1. As shown, the macrocyclic peptides ofthe present disclosure demonstrated potent inhibition of PD-1-Ig bindingactivity to PD-L1-TVMV-His (PD-L1-His). Ranges are as follows:A=0.10-1.6 μM; B=0.01-0.099 μM; C=0.0003-0.0099 μM.

TABLE 1 Example Number HTRF IC50 (μM) INT 1300B C 14089 C 14090 C  3214C  3619 C  3620 0.0090  3621 B  3622 B  3623 C  3624 C  3625 0.0063 3626 C INT 1300C C INT 1400K C INT 1400L B 11012 B 11032 0.2007 11033 B11035 B 11036 C 11040 C 11041 C 11042 0.0049 11044 B 11045 B 11046 B11047 B 11060 A 11061 A 11062 0.4558 11064 C 11066 C 11067 B 11073 B11074 C 11075 0.0071 11076 B 11080 0.0127 11081 B 11082 B 11083 B 11085B 11086 0.0190 11087 B 11088 A 11089 B 11090 B 11102 0.0130 11115 A11119 A 11129 B 11130 B 11131 B 11132 B 11133 1.5710 11013 B 11015 B11028 B 11029 A 11034 C 11038 C 11063 0.0092 11065 B 11068 C 11072 C11077 C 11084 B 11112 A 11124 A 11125 B 11128 B 11017 0.2593 11018 B11071 C 11078 C 11101 0.0041 11104 A 11107 A 11109 A 11069 B 11070 C11079 B 11091 C 11092 0.0135 11093 B 11094 0.0188 11095 C 11096 B 11097B 11098 B 11099 C 11100 B 11110 0.3140 11111 A 11108 A 11120 A INT 1300VB INT 1300W B INT 1300X B INT 1300Y C 11001 0.9522 11019 A INT 1300A CINT 1400A C INT 1400B C INT 1400C C INT 1400D C INT 1400E C INT 1400F CINT 1400G C INT 1400H C INT 1400I B INT 1400J 0.0088 11002 B 11007 B11008 A 11020 A 11031 B 11134 B 11005 B 11006 B 11009 0.0914 11010 B11011 B 11016 B 11014 A 11030 A 11103 B 11116 B 11123 B 11126 B 110030.0546 11004 B 11021 A 11022 A 11023 A 11024 A 11025 A 11026 A 11027 A11114 A 11135 B 11136 B 11137 B 11138 B 11139 B 11140 B 11141 B 11142 B11143 C 11144 B 11145 C 11146 B 11147 V 11148 V 11149 V 11150 B 11151 B11152 A 11153 B 11154 C 11155 C 11156 C 11157 B 11158 B 11159 B 11160 A11161 A 11162 A 11163 C 11164 C 11165 C 11166 A 11167 C 11168 C 111690.0039 11170 C 11171 C 11172 B 11173 A 11174 B 11175 B 11176 B 11177 A11178 C 11179 0.003  11180 B 11181 A 11182 A 11183 A 11184 B 11185 B11186 — 11187 B 11188 0.28  11189 A 11190 B 11191 B 11192 B 11193 B11194 B 11195 B 11196 B 11197 B 11198 B 11199 B 11200 B 11201 B 11202 B11203 B 11204 B 11205 0.01  11206 B 11207 B 11208 B 11209 B 11210 B11211 C 11212 B 11213 B 11214 B 11215 B 11216 B 11217 B 11218 B 11219 B11220 B 11221 B 11222 B 11223 B 11224 C 11225 C 11226 C 11227 C 11228 B11229 B 11230 A 11231 B 11232 B 11233 C 11234 C 11235 B 11236 C 11237 B11238 C 11239 B 11240 0.01  11241 B 11242 B 11243 A 11244 A 11245 B11246 B 11247 B 11248 0.22  11249 C 11250 C 11251 B 11252 B 11253 C11254 C 11255 C 11256 B 14051 A 14052 B 14053 A 14054 A 14055 0.388014056 A 14057 A 14058 A 14060 A 14085 B 14086 B 14087 A 14088 0.798914082 B INT130AA B INT130AB B INT130AF C INT130AG C INT130AI C INT130ADB INT130AE B INT130AH C INT130AJ C INT130AK C INT130AL C 13051 B 13052 B13122 A 13123 0.150  13124 B 13125 B 13126 B 13127 B 13128 0.013  13129B 13130 C 13131 B 13132 0.0033 13133 C 14059 B 14061 B 14062 B 14063 B14064 C 14065 B 14066 C 14067 0.0104 14068 B 14069 B 14070 B 14071 B14072 0.0085 14073 B 14074 B 14075 B 14076 C 14077 0.0209 14078 B 14079B 14080 B 14081 A 14083 B 14084 0.6270 14092 C 14093 C 14094 B 140950.014  14096 0.790  14068 B 14097 0.0072 14098 C 14099 C 14100 C 141010.0062 14069 B 14070 B 14071 B 14072 0.0085 14073 B 14074 B 14075 B14076 C 14077 B 14078 0.0108 14079 B 14080 B 14081 A 14083 B 140840.6270 14092 C 14102/14103 0.014  14104 A 14105 A 14106 A 14107 A 14121B 14122 B 13141 A 13142 A 13143 A 14123 C 13144 B 13145 B 14124 B 13146B 13147 A 13148 B 13149 B 13150 0.0209 13151 B 13152 C 13153 C 13154 C13155 C 13156 C 13157 C 13158 C 13159 C 13160 C 13161 C 13162 0.003813163 C 13164 C 14125 C 14126 C 11257 C 11258 B 11259 C 11260 C 11261 C11262 B 11263 C 11264 C 11265 B 11266 C 11267 B 11268 A 11269 ND 11270ND 11271 ND

It will be evident to one skilled in the art that the present disclosureis not limited to the foregoing illustrative examples, and that it canbe embodied in other specific forms without departing from the essentialattributes thereof. It is therefore desired that the examples beconsidered in all respects as illustrative and not restrictive,reference being made to the appended claims, rather than to theforegoing examples, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

1.-37. (canceled)
 38. A method of enhancing, stimulating, and/orincreasing an immune response in a subject in need thereof, said methodcomprising administering to the subject a therapeutically effectiveamount of a compound of formula (I)

or a pharmaceutically acceptable salt thereof, wherein: A is

wherein:

denotes the point of attachment to the carbonyl group and

denotes the point of attachment to the nitrogen atom; m is 1; w is 0;R¹⁴ and R¹⁵ are hydrogen; R^(16a) is hydrogen; R¹⁶ is—(C(R^(17a))₂)₂—X—R³⁰, X is a chain of between 8 and 46 atoms whereinthe atoms are selected from carbon and oxygen and wherein the chain maycontain one, two, or three C(O)NH groups embedded therein; and whereinthe chain is optionally substituted with one or two groups independentlyselected from —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and —CH₂CO₂H; R³⁰ isselected from —CO₂H, —C(O)NR^(w)R^(x), and —CH₃ wherein R^(w) and R^(x)are hydrogen, provided that when X is all carbon, R³⁰ is other than—CH₃; each R^(17a) is hydrogen, each of R^(c), R^(f), R^(h), R^(i),R^(m), and R^(n) is hydrogen; R^(a), R^(e), R^(j), and R^(k), are eachindependently selected from hydrogen and methyl; R¹ is methyl; R¹ isphenylC₁-C₃alkyl wherein the phenyl part is optionally substituted withhydroxyl, halo, or methoxy; R² is C₁-C₇alkyl and R^(b) is methyl; or, R²and R^(b), together with the atoms to which they are attached, form apiperidine ring; R³ is NR^(x)R^(y)(C₁-C₇alkyl),NR^(u)R^(v)carbonylC₁-C₃alkyl, or carboxyC₁-C₃alkyl; R⁴ and R^(d),together with the atoms to which they are attached, form a pyrrolidinering; R⁵ is hydroxyC₁-C₃alkyl, imidazolylC₁-C₃alkyl, orNR^(x)R^(y)(C₁-C₇alkyl); R⁶ is carboxyC₁-C₃alkyl,NR^(u)R^(v)carbonylC₁-C₃alkyl, NR^(x)R^(y)(C₁-C₇alkyl), or C₁-C₇alkyl;R⁷ and R^(g), together with the atoms to which they are attached, form apyrrolidine ring optionally substituted with hydroxy; R⁸ and R¹⁰ arebenzothienyl or indolylC₁-C₃alkyl optionally substituted withcarboxyC₁-C₃alkyl; R⁹ is hydroxyC₁-C₃alkyl, aminoC₁-C₃alkyl, orC₁-C₇alkyl; R¹¹ is C₁-C₃alkoxyC₁-C₃alkyl or C₁-C₇alkyl; R¹ is C₁-C₇alkylor hydroxyC₁-C₃alkyl; and R¹³ is C₁-C₇ alkyl, carboxyC₁-C₃alkyl, or—(CH₂)₃NHC(NH)NH₂.
 39. The method of claim 38, further comprisingadministering an additional agent prior to, after, or simultaneouslywith the compound of formula (I), or a therapeutically acceptable saltthereof.
 40. The method of claim 39, wherein the additional agent is anantimicrobial agent, an antiviral agent, a cytotoxic agent, and/or animmune response modifier.
 41. The method of claim 38, wherein thecompound of formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.
 42. A method ofinhibiting growth, proliferation, or metastasis of cancer cells in asubject in need thereof, said method comprising administering to thesubject a therapeutically effective amount a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein: A is

wherein:

denotes the point of attachment to the carbonyl group and

denotes the point of attachment to the nitrogen atom; m is 1; w is 0;R¹⁴ and R¹⁵ are hydrogen; R^(16a) is hydrogen; R¹⁶ is—(C(R^(17a))₂)₂—X—R³⁰, X is a chain of between 8 and 46 atoms whereinthe atoms are selected from carbon and oxygen and wherein the chain maycontain one, two, or three C(O)NH groups embedded therein; and whereinthe chain is optionally substituted with one or two groups independentlyselected from —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and —CH₂CO₂H; R³⁰ isselected from —CO₂H, —C(O)NR^(w)R^(x), and —CH₃ wherein R^(w) and R^(x)are hydrogen, provided that when X is all carbon, R³⁰ is other than—CH₃; each R^(17a) is hydrogen, each of R^(c), R^(f), R^(h), R^(i),R^(m), and R^(n) is hydrogen; R^(a), R^(e), R^(j), and R^(k), are eachindependently selected from hydrogen and methyl; R¹ is methyl; R¹ isphenylC₁-C₃alkyl wherein the phenyl part is optionally substituted withhydroxyl, halo, or methoxy; R² is C₁-C₇alkyl and R^(b) is methyl; or, R²and R^(b), together with the atoms to which they are attached, form apiperidine ring; R³ is NR^(x)R^(y)(C₁-C₇alkyl),NR^(u)R^(v)carbonylC₁-C₃alkyl, or carboxyC₁-C₃alkyl; R⁴ and R^(d),together with the atoms to which they are attached, form a pyrrolidinering; R⁵ is hydroxyC₁-C₃alkyl, imidazolylC₁-C₃alkyl, orNR^(x)R^(y)(C₁-C₇alkyl); R⁶ is carboxyC₁-C₃alkyl,NR^(u)R^(v)carbonylC₁-C₃alkyl, NR^(x)R^(y)(C₁-C₇alkyl), or C₁-C₇alkyl;R⁷ and R^(g), together with the atoms to which they are attached, form apyrrolidine ring optionally substituted with hydroxy; R⁸ and R¹⁰ arebenzothienyl or indolylC₁-C₃alkyl optionally substituted withcarboxyC₁-C₃alkyl; R⁹ is hydroxyC₁-C₃alkyl, aminoC₁-C₃alkyl, orC₁-C₇alkyl; R¹¹ is C₁-C₃alkoxyC₁-C₃alkyl or C₁-C₇alkyl; R¹² isC₁-C₇alkyl or hydroxyC₁-C₃alkyl; and R¹³ is C₁-C₇ alkyl,carboxyC₁-C₃alkyl, or —(CH₂)₃NHC(NH)NH₂.
 43. The method of claim 42,wherein the cancer is selected from melanoma, renal cell carcinoma,squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC,colorectal cancer, castration-resistant prostate cancer, ovarian cancer,gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, squamouscell carcinoma of the head and neck, carcinomas of the esophagus,gastrointestinal tract and breast, and hematological malignancies. 44.The method of claim 42, wherein the compound of formula (I) is selectedfrom:

or a pharmaceutically acceptable salt thereof.
 45. A method of blockingthe interaction of PD-L1 with PD-1 and/or CD80 in a subject in needthereof, said method comprising administering to the subject atherapeutically effective amount of a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein: A is

wherein:

denotes the point of attachment to the carbonyl group and

denotes the point of attachment to the nitrogen atom; m is 1; w is 0;R¹⁴ and R¹⁵ are hydrogen; R^(16a) is hydrogen; R¹⁶ is—(C(R^(17a))₂)₂—X—R³⁰, X is a chain of between 8 and 46 atoms whereinthe atoms are selected from carbon and oxygen and wherein the chain maycontain one, two, or three C(O)NH groups embedded therein; and whereinthe chain is optionally substituted with one or two groups independentlyselected from —CO₂H, —C(O)NH₂, —CH₂C(O)NH₂, and —CH₂CO₂H; R³⁰ isselected from —CO₂H, —C(O)NR^(w)R^(x), and —CH₃ wherein R^(w) and R^(x)are hydrogen, provided that when X is all carbon, R³⁰ is other than—CH₃; each R^(17a) is hydrogen, each of R^(c), R^(f), R^(h), R^(i),R^(m), and R^(n) is hydrogen; R^(a), R^(e), R^(j), and R^(k), are eachindependently selected from hydrogen and methyl; R¹ is methyl; R¹ isphenylC₁-C₃alkyl wherein the phenyl part is optionally substituted withhydroxyl, halo, or methoxy; R² is C₁-C₇alkyl and R^(b) is methyl; or, R²and R^(b), together with the atoms to which they are attached, form apiperidine ring; R³ is NR^(x)R^(y)(C₁-C₇alkyl),NR^(u)R^(v)carbonylC₁-C₃alkyl, or carboxyC₁-C₃alkyl; R⁴ and R^(d),together with the atoms to which they are attached, form a pyrrolidinering; R⁵ is hydroxyC₁-C₃alkyl, imidazolylC₁-C₃alkyl, orNR^(x)R^(y)(C₁-C₇alkyl); R⁶ is carboxyC₁-C₃alkyl,NR^(u)R^(v)carbonylC₁-C₃alkyl, NR^(x)R^(y)(C₁-C₇alkyl), or C₁-C₇alkyl;R⁷ and R^(g), together with the atoms to which they are attached, form apyrrolidine ring optionally substituted with hydroxy; R⁸ and R¹⁰ arebenzothienyl or indolylC₁-C₃alkyl optionally substituted withcarboxyC₁-C₃alkyl; R⁹ is hydroxyC₁-C₃alkyl, aminoC₁-C₃alkyl, orC₁-C₇alkyl; R¹¹ is C₁-C₃alkoxyC₁-C₃alkyl or C₁-C₇alkyl; R¹² isC₁-C₇alkyl or hydroxyC₁-C₃alkyl; and R¹³ is C₁-C₇ alkyl,carboxyC₁-C₃alkyl, or —(CH₂)₃NHC(NH)NH₂.
 46. The method of claim 45,wherein the compound of formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.